Porous silica substrates for polymer synthesis and assays

ABSTRACT

Methods are provided for making and using thin films of porous silica substrates to synthesize arrays of polymers. Methods are also provided for assaying such polymers on porous silica substrates. The porous silica substrates offer an increase in array density and signal enhancement over conventional flat glass substrates. Examples of polymers that can be synthesized and assayed include biological polymers such as nucleic acids, polynucleotides, polypeptides, and polysaccharides. Arrays of nucleic acids or polynucleotides can be used for a variety of hybridization-based experiments such as nucleic acid sequence analysis, nucleic acid expression monitoring, nucleic acid mutation detection, speciation, effects of drug therapy on nucleic acid expression, among others.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication Serial No. 60/128,402, filed Apr. 8, 1999, the disclosure ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] This invention pertains to preparation and use of very highsurface area porous substrates that can be used to synthesize highdensity arrays of polymers.

BACKGROUND

[0003] Porous silica glass has been known for quite some time. U.S. Pat.No. 4,220,461 provides a historical perspective and discussion on thedevelopment of silica-rich phase-separable porous glass. Various methodsfor the manufacture of phase-separable porous glass are reviewed in U.S.Pat. No. 4,528,010. Both of these references are incorporated byreference in their entireties for all purposes.

BRIEF DESCRIPTION OF THE INVENTION

[0004] The present invention relates to a porous substrate and methodsfor making and using the porous substrate. The porous substrate providesan increased surface area for polymers to attach to the substrate. Suchporous substrates are often used to make an array of polymers, such asfor genetic diagnostic purposes. The polymers may be placed orfabricated on the porous substrate by various methods.

[0005] The polymers can include those of biological interest such asnucleic acids, polynucleotides, proteins, polypeptides, polysaccharides,oligosaccharides, mixtures of these or other polymers on an array andcombinations of the above polymer units in individual polymers. Theporous substrates thus are useful in, for example, glass technology,polymer chemistry, molecular biology, medicine, and medical diagnostics.

[0006] The porous substrate generally has at least two regions, asupport region and a porous region. The support region, which can serveas an underlayer region, basically provides mechanical support for easeof handling of a porous region. The porous region may be for example alayer (film). The support region can be selected or processed to provideadditional features in the finished porous substrate. One advantage ofusing a porous region with higher surface area to make an array is thatthe array can be functionalized with a much higher density of polymersfor a given two dimensional area without changing the spacing betweenpolymers on the surface of the porous substrate.

[0007] One embodiment of this invention provides a primarily inorganicporous substrate including a support region, and a porous region incontact with the support region. The porous region for example includespores with a pore size of 1-500 nm, or 2-500 nm, the porous regionhaving a porosity of, e.g., 10-90%, 20-80%, or 70-90%, and a poroussurface thickness of 0.01-20 μm, wherein the porous region has a surfacecapable of forming arrays of polymers thereon. The porosity isgenerally, for example, “open”, that is, some pores are connected toothers to allow the infusion of polymers or other fluids. Not all thepores need to connect to another, that is, some of the pores may beclosed. What is meant by “primarily inorganic” is that a small amount oforganic material may remain in the porous region of the substrate, ormay be intentionally applied onto the surface(s) of the porous region.

[0008] In one embodiment, a porous substrate is provided comprising:

[0009] a support region; and

[0010] a porous region on the support region, the porous region beingprimarily inorganic and having a surface capable of forming a polymerarray thereon, the porous region comprising pores of a pore size ofabout 2 nm-500 nm or 1000 Angstroms to 500 nm, a porosity of about10-90%, and a thickness of about 0.01 μm to about 70 μm.

[0011] The porous region can be formed by an additive method, which caninclude the application of colloidal silica on the support region. Theadditive method also may include the application of alkoxysilane on thesupport region. The porous region may comprise silica. The porous regionmay further comprise organic polymer of less than or equal to about 10%mole fraction. The porous region may comprise a plurality of pores, eachof the plurality of pores having a size of from about 2 to about 100 nm.The porous region may comprise a plurality of pores, each of theplurality of pores having a size of from about 2 to about 50 nm. Theporous region has, for example, a porosity of from about 20 -80%, or50-70%. The porous region for example comprises a plurality ofparticles, each of the plurality of particles having a size from about5-500 nm, 5-200 nm, or 70-100 nm. The porous region has, for example, athickness from about 0.1-1 microns, or about 0.1 μm to about 0.5 μm, orabout 1 μm to about 20 μm.

[0012] An organic polymer may coat silica particles of the porousregion. The porous region may be silylated with a silyating agent, suchas N,N-bis(hydroxyethylaminopropyl)triethoxysilane and glycidoxypropyltrimethoxy silane. The porous region may be formed by codepositing anorganic template material with silica, followed by removing the organictemplate material. The organic template material for example comprisesparticles of about 10-100 nm and the silica comprises particles of about7-100 nm. The organic template particle size can be about equal to asilica particle size. The silica particle size is for example less thanor equal to about ⅔ an organic template particle size. The silicaparticle size is in one embodiment, less than about 10% of an organictemplate particle size. The organic template material can be depositedin a volume ratio to the silica of about 10:1 to 1:10, e.g., 2:1. Theorganic template material is in one embodiment removed using a bakingprocess at a temperature of above about 150° C. The silica may bedensified using an annealing process. The porous region has in oneembodiment an effective surface area about 15-40 times a flat substratewith an equivalent two dimensional structure. In one embodiment, theporous region is formed by a subtractive method. The organic templatepolymer may be a latex polymer. The porous substrate may comprisephase-separable glass, a surface portion of the phase-separable glassbeing treated to form the porous layer. The phase-separable glass maycomprise for example a sodium borosilicate glass. The sodiumborosilicate glass may be been annealed and leached to provide theporous layer having a thickness of about 70 microns and comprised of aplurality of pores, at least some of the plurality of pores having apore size greater than about 1000 Å. The porous region has, e.g., aneffective surface area about 50-400 times a flat substrate with anequivalent two dimensional structure.

[0013] The porous substrate may further comprise a high density array ofpolymers, such as nucleic acids immobilized on the surface.

[0014] In another embodiment, a porous substrate is provided comprising:

[0015] a support region; and

[0016] a porous region on the support region, said porous region beingabout 0.1-0.5 microns thick,

[0017] wherein the porous layer comprises an unsintered matrix formedfrom at least colloidal silica having a particle size of about 70-100microns, the unsintered matrix defining at least a plurality of openpores having a pore size of about 10-20 nm, and

[0018] wherein the porous layer has a porosity of of about 10-90%.

[0019] In one embodiment, a method of forming a porous substrate isprovided, the method comprising:

[0020] providing a substrate material comprising a surface;

[0021] dipping the substrate material in a solution including colloidalsilica and a carrier, the colloidal silica having a particle size ofabout 12-100 nm; and

[0022] withdrawing the substrate material to provide an unsinteredporous layer having a thickness of about 0.1-1 microns and a porosity ofof about 10-90% on the substrate material.

[0023] Also provided is a method of forming a porous substrate, themethod comprising:

[0024] providing a substrate material comprising a surface;

[0025] applying a solution including colloidal silica and a carrier tothe surface of the substrate material, the colloidal silica having aparticle size of about 12-100 nm;

[0026] spinning the substrate material and the applied solution toachieve a spun layer on the substrate material; and

[0027] removing the carrier from the spun layer to provide an unsinteredporous layer having a thickness of about 0.1-1 microns and a porosity ofabout 10-90% on the substrate material.

[0028] Another embodiment is a method of forming a porous substratecomprising different monomer sequences, the method comprising:

[0029] immobilizing different monomer sequences on a porous substrate.

[0030] In another embodiment, there is provided a method of synthesizingpolymers on a porous substrate, the method comprising:

[0031] a) generating a pattern of light and dark areas by selectivelyirradiating at least a first area of a surface of a porous substrate,said surface comprising immobilized monomers on said surface, saidmonomers coupled to a photoremovable protective group, withoutirradiating at least a second area of said surface, to remove saidprotective group from said monomers in said first area;

[0032] b) simultaneously contacting said first area and said second areaof said surface with a first monomer to couple said first monomer tosaid immobilized monomers in said first area, and not in said secondarea, said first monomer having said photoremovable protective group;

[0033] c) generating another pattern of light and dark areas byselectively irradiating with light at least a part of said first area ofsaid surface and at least a part of said second area to remove saidprotective group in said at least a part of said first area and said atleast a part of said second area;

[0034] d) simultaneously contacting said first area and said second areaof said surface with a second monomer to couple said second monomer tosaid immobilized monomers in at least a part of said first area and atleast a part of said second area; and

[0035] e) performing additional irradiating and monomer contacting andcoupling steps so that a matrix array of different polymers is formed onsaid surface, whereby said different polymers have sequences andlocations on said surface defined by the patterns of light and darkareas formed during the irradiating steps and the monomers coupled insaid contacting steps.

[0036] The monomers are for example, nucleotides, amino acids, ormonosaccharides. The substrate may have linker molecules on its surface.

[0037] There also is provided a method of forming polymers havingdifferent monomer sequences on a porous substrate, the methodcomprising:

[0038] providing a porous substrate comprising a linker molecule layerthereon, said linker molecule layer comprising a linker molecule and aprotective group;

[0039] applying a barrier layer overlying said linker molecule layer,said applying step forming selected exposed regions of said linkermolecule layer;

[0040] exposing said selected exposed regions of said linker moleculelayer to a deprotecting agent to remove the protective group; and

[0041] coupling selected monomers to form selected polymers on thesubstrate.

[0042] The deprotection agent may be, for example, in the vapor phase orliquid phase, and may be, for example an acid, such as trichloroaceticacid, dichloroacetic acid, or HCl. The monomers are for examplenucleotides, amino acids, or monosaccharides.

[0043] In another embodiment, there is provided a method for detecting anucleic acid sequence, the method comprising:

[0044] (a) providing an array of nucleic acids bound to the poroussubstrate;

[0045] (b) contacting the array of nucleic acids with at least onelabeled nucleic acid comprising a sequence substantially complementaryto a nucleic acid of said array, and

[0046] (c) detecting hybridization at least the labeled complementarynucleic acid to nucleic acids of said array.

[0047] In one embodiment, the porous substrates comprising arrays may beused to screen for a previously identified polymorphic variant in atarget nucleic acid sequence, or for a target such as a humanimmunodeficiency virus sequence. Nucleic acids such as a p53 gene, anHIV RT gene, a CFTR gene, or a cytochrome p450 gene can be screened for.The array may include, for example, at least 3200 polynucleotide probes,or, e.g., at least 10,000 polynucleotide probes, or at least 50,000probes. The probes may be, for example, 9 to 21 nucleotides in length.

BRIEF DESCRIPTION OF FIGURES

[0048]FIG. 1 is a simplified cross section of a portion of a poroussubstrate with a porous region formed from particles according to oneembodiment of the invention.

[0049]FIG. 2 is a simplified cross section of a portion of a poroussubstrate with a porous region formed by leaching according to oneembodiment of the invention.

[0050]FIG. 3a is a simplified cross section of a portion of a substratein an intermediate processing state with templating particles that aresubstantially larger than interstitial silica particles according to oneembodiment of the invention.

[0051]FIG. 3b is a simplified cross section of the portion of thesubstrate shown in FIG. 3a after the templating material has beenremoved to form a porous substrate.

[0052]FIG. 4 is a simplified cross section of a portion of a substratein an intermediate processing state according to another embodiment ofthe present invention with templating particles of about the same sizeas silica particles.

DESCRIPTION OF THE INVENTION

[0053] The present invention relies on many patents, applications andother references for details known to those of the art. Therefore, whena patent, application or other reference is cited or repeated below, itshould be understood that it is incorporated by reference in itsentirety for all purposes as well as for the proposition that isrecited.

[0054] The present invention provides a porous substrate and methods formaking and using the porous substrate. The porous substrate provides alarge surface area for polymers to be attached to make an array. Thepolymers may be placed or fabricated on the array by various methods. Aporous layer is formed on a substrate material, and in some embodiments,the porosity, pore size, and thickness of the porous layer is chosenaccording to desired functionalization characteristics. Poroussubstrates are generated by creating a 3D matrix to increase the surfacearea and therefore increase the number of sites available for arraysynthesis in the same lateral dimensions. One advantage in using aporous layer to increase the effective surface area is to make an arraythat can be functionalized with a much higher density of polymers for agiven two dimensional, or “flat” area without changing the spacingbetween cells of the array on the surface of the substrate. Theeffective surface area is the surface area of the porous region that isavailable for adsorption of polymer molecules or for polymer synthesis,of example.

[0055] The support region can be, for example sodalime glass, borafloatglass, sodium borosilicate glass, fused silica, or a polymer, such asplastic. When the porous layer is silica, it can be manufactured by manymeans. Two exemplary ways to form the porous region are by the additionof material (e.g. deposition), and by removal of material (e.g.selective etching).

[0056] In additive methods, a porous region is formed on the surface ofthe underlying substrate to increase the effective surface area. Theporous region can be formed from deposition of any or all of thefollowing with or without catalysts in appropriate solvent and ratios.For example, the porous region can be formed from colloidal silica, anorgano-silicon compound, such as tetramethoxysilane (TMOS), metalalkoxides, silsesquioxanes, or other silanes, or-combinations of thesematerials typically used in sol gel processes. See C. J. Brinker,Sol-Gel Science, Academic Press, Boston, 1990. With these types ofprecursors, parameters such as solution composition, concentration, pH,aging time, and temperature can be used to tailor the morphology (poresize, porosity, thickness) of the porous region that is formed.Additionally, there can be combinations of the above techniques toprovide the same eventual result (and there can be combinations in theadditive and subtractive techniques to achieve similar results for otherpurposes). Also, other inorganic materials can be used in either of thesame forms as above (such as aluminum or titanium-based materials).

[0057] Furthermore, the porous region matrix can be “templated”. In atemplating process, a sacrificial material, such as a polymer, isdeposited with the matrix and then burned out, leaving behind a porousstructure with selected characteristics. (Note that a porous region canalso be formed without templating). The template material can be any ofthe following, a preformed polymer, such as a polystyrene latex,polymers dissolved in solution, or a combination of these materials. Thebum-out process, typically done by heating in air, can be carried out attemperatures above 150° C. up to the melting point (or glass transitiontemperature, if appropriate) of the material that will form the matrixfor the porous layer. After the templating material is burned out, thematrix material can be sintered together. Those skilled in the art willappreciate that the term “sintering” (or “annealing” in some contexts)is used to describe a time-temperature processes for heating glass orother particles to cause them to join. Whether the process is strictlysolid state, or involves some amount of material liquefaction orsoftening, is not essential if a porous structure results. Time andtemperature of the sintering process can be varied to achieve differentamounts of densification and pore characteristics. Post-treatments afterannealing can be used to clean the surface in preparation for arraysynthesis, such as cleaning in a solution of sulfuric acid and hydrogenperoxide (“piranha solution”) or sodium hydroxide. The porous regionmatrix particles and templating particles can be applied to the surfaceof the underlying substrate individually, or as a pre-agglomerated mass.

[0058] A porous region, such as a porous layer or film can be formed ona surface of the underlying substrate by a variety of processes, such asspin-coating, dip-coating, spraying (aerosol), individual spotsdeposited on surface, use of barriers (physical or chemical) tospecifically deposit coatings into channels, pads, spots, or patternedsurfaces. It should be understood that the porous region or layer can becreated in various forms, shapes, or areas, or over the entire surface.

[0059] In preferred embodiments, film thickness can be modulated byaltering either or both of the precursors or layer formation conditions.For example, the weight percent of solids in the reagents listed above(e.g., about 10 wt. % to 40 wt. % solution of colloidal silica) canalter the layer thickness. Similarly, performing multiple depositionscan build up the layer thickness. Additional processing might be donebetween application of successive layers of reagents, such as baking thesubstrate to remove solvents from the film before the next applicationof reagents. Film thickness can also be altered by modulating the spinor deposition speed, for example, slower speeds yielding thicker filmsand faster speeds yielding thinner films. Also, altering the pull speedout of the dip-coating bath can affect layer thickness, as slower speedsyield thinner films and faster speeds yield thicker films. One can alsocontrol the solution conditions to affect the film thickness. Forexample, when using the TMOS approach, the solution can be caused tobegin gelling, which increases the viscosity and therefore the thicknessof the deposited layer.

[0060] The coatings, particles or other components can be spun onto thesubstrate surface, the substrate can be dipped in a solution containingthe above reagents, the reagents can be sprayed onto the surface of thesubstrate, or applied by other methods. The substrate can be treated tocreate areas of high porosity over the entire surface of the substrateor just in select locations, such as by spotting reagents in grids,circular spots, areas, cells or any shape that is preferred (see U.S.Pat. Nos. 5,744,305; 5,445,934; and 6,040,138). When the porosity ishigh and the pores or particles are small, it minimizes the lightscattering properties when read by an instrument that uses opticalproperties to detect a reaction (see U.S. Pat. Nos. 5,744,305;5,981,956; and 6,025,601).

[0061] A subtractive approach can also be used to increase the porosityof the substrate. For example, a porous region can be etched into thesurface of the substrate (i.e. the material at the surface of what willbecome the underlying substrate). The surface can be prepared as etchedglass, such as phase-separating sodium borosilicate glass, techniquesfor which are known in the art. In a particular embodiment of thesubtractive approach to forming a porous substrate, the pore size can befurther controlled by the annealing time and temperature of an annealingstep performed before the etch (longer annealing, higher temperaturesincrease pore size). Also, the depth of the porous region can becontrolled by etching parameters (solution concentration, composition,time, etc.) in accordance with the substrate material.

[0062] In some embodiments, the silica substrate has an organic polymercontent of less than or equal to 10% mole fraction. In some embodiments,an organic polymer coats the porous substrate. In some embodiments, theporous substrate is coated with a silane compound capable of linkingwith a polymer, such as glycidoxypropyl-trimethoxysilane or withN,N-bis(hydroxyethylaminopropyl)triethoxysilane.

[0063] In either the subtractive or additive techniques, it is desirableto increase the effective surface area of the underlying substrate sothat more polymers can be attached to the surface. In one embodiment,the pore size is at least 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 200,250, or 500 nanometers. In one embodiment, the largest pore size is 750,800, 900, or 1,000 nanometers. It is understood that not all pores willbe of precisely the same size, but rather will fall within a range in agiven porous layer. The numbers used are merely examples of theapproximate average pore diameter. The effective surface area can beexpressed in units of meters of surface area per gram of layerdeposited. In one embodiment, at least 15, 20, 25, 30, 50, 75, 100, 200,250, 500 meters of surface area is per gram of layer deposited is formedand not more than 750, 800, 900, or 1,000 meters of surface area pergram of layer deposited.

[0064] The effective surface area can also be expressed as a factor ofarea enhancement over the flat surface area of the underlying substrate.For example, if the flat surface area is expressed as “1”, the increasein the effective surface area is for example at least 2, 5, 10, 15, 20,25, 30, 50, 75, 100, 200, 250, 500, 750, or 1,000 times the flat surfacearea. Porosity is for example expressed as at least 10, 20, 30, 40, 50,60, 70, 80 or 90% of the surface of the substrate actually treated. Notethat when a film is deposited on a support material, the “porosity”refers to the porosity of the film. The porosity of the layer is definedas the space occupied by void divided by the sum of the space occupiedby both solid material and void within the layer. The array may containareas that have not been treated to increase the surface area or thewhole surface can be treated, or selected areas might be treateddifferently. A porous region formed by depositing colloidal silica forexample has a thickness of about 0.01-20 microns, or about 0.1-1.0microns, about 0.1 -0.5 microns, or about 0.1 to 10 microns. It wasfound that this layer thickness provided good surface area enhancementfor processes such as bioassay and biosynthesis, and could be reliablyfabricated according to methods of the present invention.

[0065] In a preferred embodiment, the present invention is a poroussilica substrate that is useful for the manufacture of an array ofpolymers. Polymer arrays and their uses are well described in manypatents and applications. Example uses of polymer arrays include geneexpression monitoring, discovery of polymorphisms, genotyping,diagnostics, and affinity columns. See the following, which areincorporated by reference in their entireties for all purposes. See U.S.Pat. Nos. 5,677,195, 5,631,734, 5,624,711, 5,599,695, 5,510,270,5,445,934, 5,451,683, 5,424,186, 5,412,087, 5,405,783, 5,384,261,5,252,743 and 5,143,854; and U.S. application Ser. No. 08/388,321, filedFeb. 14, 1995, the disclosures of each of which are incorporated herein.Uses of these polymer arrays are described in U.S. Pat. Nos. 5,795,7165,800,992, and 6,040,138.

[0066] Porous silica has been produced for other uses, such as anintermediate form of a silica glass article. A process according to thepresent invention utilizes a similar removal method with aphase-separable glass body (substrate) in combination with an etchingprocess and possibly post-etch thermal treatment(s) to form a porouslayer on the underlying substrate. The removal method involves (1)forming an article of desired shape from a parent sodium borosilicateglass; (2) thermally treating the glass article at a temperature of500-600° C. to separate the glass into a silica-rich phase and asilica-poor phase; (3) dissolving or leaching the silica-poor phase withacid to provide a porous structure composed of the silica-rich phase;and (4) washing to remove leaching residue, and then drying.

[0067] Another method according to the present invention uses “sol-gel”in a deposition process to prepare a porous substrate at moderatetemperatures. Production of porous inorganic oxide glass by the sol-gelprocess are described in U.S. Pat. Nos. 3,640,093 and 4,426,216. Seealso Scholze et al., J. Non-Crystalline Solids, 73: 669 (1985). Thesol-gel procedure involves the formation of a three-dimensional networkof metal oxide bonds at room temperature by a hydrolysis-condensationpolymerization reaction of metal alkoxides, followed by low temperaturedehydration. The resultant porous glass structure optionally can besintered at elevated temperatures. More recently, U.S. Pat. No.4,765,818 described the sol-gel preparation of microporous glassmonoliths having 0.1-2 moles of trioxane per mole of tetraalkoxysilane,and reported that the glass displayed superior optical properties.

[0068] Advantages of creating porous films through sol-gel processinginclude that the films can be deposited and processed easily, are inertto most chemicals, and can be created with a wide range of morphologyand surface chemistry.

[0069] Methods of synthesis of arrays of polymers, each polymercomprising a plurality of monomers, are described in U.S. Pat. No.5,744,305 ('305) or 5,831,070. “Monomer” may be, for example, a memberof the set of individual molecules which can be joined together to forma larger polymer. Monomers can include individual units of a polymer(such as one nucleotide) or can be larger individual units (such asdimers, trimers, and higher) to make up a larger polymer by sequentialaddition of these larger units. Polymers of all types include analogousor mimics of the natural polymer units. Predefined or known region meansa localized area on a surface that contains a polymer. The region mayhave a convenient shape, e.g., circular, rectangular, elliptical,wedge-shaped, etc. For the sake of brevity herein, “predefined or knownregions” are sometimes referred to simply as “regions.” Many synthesismethods can be used to apply polymers to these regions. These regionsare the sized as shown in '305 and 5,445,934 ('934). Exemplary regionsizes are between 1, 5, 10, 20, 25, 30, 40 and 50 microns square.Densities of the regions per square centimeter are shown in '305 and'934. For example, there are 10, 50, 100, 200, 400, 500, 700, 10³, 10⁴,10⁵, 10⁶, and 10⁷ different regions per square centimeter. Primarily inone embodiment refers to about 90% of the adjective it modifies. Thus,in one embodiment, a “primarily inorganic substrate” means a substratethat has about 90% inorganic component such as silica. The remainingportion of the substrate can have organic materials, or in some cases,trace impurities, or both. As used in the specification and claims, thesingular form “a”, “an” and “the” include plural references unless thecontext clearly dictates otherwise. For example, the term “an agent”includes a plurality of agents, including mixtures thereof.

[0070] Porous Substrate

[0071] The porous substrate is useful as an article of manufacturehaving a rigid or semi-rigid surface on which polymers can besynthesized and or various applications (such as hybridization,ligand-binding assays) using polymers can be performed. In some aspects,the porous region comprises a primarily inorganic porous materialproviding an enhanced surface area greater than the flat surface area.In some embodiments, the primarily inorganic porous region comprisessilica. In some embodiments, at least one surface of the substrate willbe substantially flat, although in some embodiments it may be desirableto physically separate synthesis regions for different polymers with,for example, wells, raised regions, etched trenches, large beads, lighttransmitting fibers, or the like. According to other embodiments, smallbeads may be provided on the surface of the substrate itself, which maybe released upon completion of the synthesis. The porous region isformed on an support material that can be of a similar or differentmaterial than the material of the porous region. Suitable materialsinclude those recited herein as support, such as all types of glassmaterials, plastics, polymers, fused silica and other rigid andsemi-rigid materials.

[0072] The porous region may be made of silica or other material ormaterials. The term “silica” represents silica compounds such as silicondioxide, although the exact stoichiometric ratio of oxygen to siliconmay vary and the silica may include modifying elements. The silica maybe in a colloidal form (which is known as colloidal silica) or in anoncolloidal form. Silica can be made from an organic compound orcompounds comprising the silicon atom such as an alkoxysilane, anexample of which is tetramethoxysilane (“TMOS”). Colloidal silica, whichis a form of very fine silica particles, can be suspended in water(commonly called a “sol”) or in an organic solvent.

[0073] The porous substrate has a porous region wherein a substantialnumber of the pores of the layer are connected to each other andeventually to the free surface of the substrate. This allows theinfusion of the porous layer with a fluid or fluids, such as a gas, aliquid, including liquid solutions, or a fluid polymer, and can providea substantial increase in surface area (compared to the flat area of thesubstrate) for molecules to attach to the surface of porous layer, aswell as providing surface area for reactions to occur. The poroussubstrate provides a three-dimensional matrix that can be functionalizedwith reactive groups, such as silylating agents, that serve as startingpoints for polymer synthesis. The porous films provide a large number ofsynthesis sites per unit area of the substrate. Additionally, the poroussubstrates hold the potential to greatly increase the binding of“target” molecules to immobilized polynucleotide or nucleic acidsequences, which would thereby enhance detection. Additionally, themultiplicity of binding sites may provide additional kineticenhancement.

[0074]FIG. 1 is a simplified cross section of a portion of a poroussubstrate 100 showing a porous region 102 formed on a support region104. The support region can be one of several different materials, suchas silica, glass, silicon, or other material that forms a suitablemechanical support for the porous region, can withstand processing, andwill not significantly effect the intended use of the substrate, such asthrough chemical reaction with assay or synthesis materials. The poroussubstrate is made up of a plurality of particles 106, 108, and 110. Inthis view, the diameters vary because of the nature of taking a crosssection, and also because there is typically some distribution ofparticle size. A free surface (“surface”) 112 has opening, or pores 114,that allow the entry of fluids, such as liquids or gas, into the porouslayer. In this example the particles are nominally 70 nm across silicaparticles. This section view represents particles of essentially thesame size that intersect the section plane. The various diameters shownin the figure represent sections of particles, some of which are notsectioned through their center. It is understood that, generally, eachparticle touches several other particles, and thus a silica matrix isformed.

[0075] Sodium Borosilicate Porous Silica Substrate

[0076] The sodium borosilicate porous silica to be used herein has thecomposition of about 65-70% SiO₂, about 24-27% B₂O₃, and about 6-8% Na₂O(by weight). In one embodiment, the composition is 67.4% SiO₂, 25.7%B₂O₃, and 6.9% Na₂O (by weight). The glass is prepared by a variety ofmethods, including by a modification of what is annealed, causing phasesto separate. The soluble phase, which is rich in sodium and boron, isthen removed by leaching with hydrochloric or hydrofluoric acid, leavingbehind a porous phase that is nearly pure silica. After leaching, athermal treatment, or anneal, can be used to modify the porousstructure. If a silica product is desired, the porous silica glass canthen be sintered at high temperatures to full density and extremely highpurity. This allows one to fabricate an article out of sodiumborosilicate glass, which has a lower softening point and is easier toform than pure silica, and end up with an article that is nearly puresilica. In the present application, the initial porosity is desired. Thepore size can be controlled by the time of annealing, while the layerdepth can be controlled by the time of leaching with acid.

[0077]FIG. 2 is a simplified cross section of a portion of a poroussubstrate 120 showing a porous region 122 formed on a support region124. The support region is generally phase separable glass, but could beother material bonded to the porous region, for example. In onepreferred embodiment, the porous region is etched in a phase separableglass that also provides the support region (i.e. the porous substrateis formed from a blank of phase separable glass). The porous region isformed by preferential leaching, as described above, and forms a“sponge-like” or “coral-like” matrix 126 with pores 128 accessible fromthe surface 130 by fluids. In contrast to the deposited porous regionshown in FIG. 1, the leached porous region has a less-defined transitionbetween the porous region 122 and the support region. It is understoodthat the figures are not to scale and are not scaled relative to eachother.

[0078] Sol-Gel Type Porous Silica Substrate

[0079] The sol-gel method can use colloidal silica with, or without analkoxysilane. A preferred embodiment uses a tetraalkoxysilane, forexample, Si(OCH₃)₄ as a starting material, which is mixed and stirredwith CH₃OH and H₂O. The resulting mixture is transferred into a desiredvessel. The vessel is allowed to stand to subject the mixture tohydrolysis and condensation reactions.

[0080] Illustrative of tetraalkoxysilanes and other metal and metalloidalkoxides that can be used in this invention are methoxy and ethoxyderivatives of silicon, lithium, sodium, potassium, rubidium, cesium,magnesium, calcium, strontium, barium, titanium, zirconium, vanadium,tantalum, chromium, molybdenum, tungsten, manganese, iron, nickel,cobalt, copper, zinc, cadmium, boron, aluminum, phosphorus, gallium,germanium, tin, arsenic, antimony, bismuth, selenium, and the like.Aryloxy derivatives such as trimethoxyphenoxysilane also can be utilizedin the sol-gel process.

[0081] Illustrative of water-miscible solvents which can be employed ina sol-gel process embodiment are alcohols such as methanol and ethanol;ketones such as acetone and methyl ethyl ketone; esters such as methylacetate and ethyl formate; ethers such as dibutyl ether andtetrahydrofuran; amides such as formamide, dimethylformamide,dimethylacetamide and 1-methyl-2-pyrrolidinone; and the like.

[0082] Acidic pH conditions in the sol-gel process can be provided bythe addition of mineral acids such as hydrochloric acid, and basic pHconditions can be provided by the addition of bases such as ammoniumhydroxide. Hydrogen fluoride is a particularly preferred acidic pHreagent, because the fluoride anions have a catalytic effect on thehydrolysis and condensation reactions of the sol-gel process.

[0083] Characteristics of the Porous Silica Substrate

[0084] In one embodiment, the porous region has the following preferredcharacteristics: a pore size of 2-500 nm, e.g., 2-100 nm, 2-200 nm, or2-50 nm; a porosity of 10-90%, e.g., 10-30%, 20-80%, 40-60%, 70-90%, or50-70%; and a thickness of 0.01-20 μm, 0.1-0.5 μm, 0.1-1.0 μm, or 1-20μm. When the substrate is prepared from a colloidal silica deposition,the average particle diameter is in one embodiment 5-500 nm, 5-100 nm,or 70-100 nm. It is well-known in the the art how to measure anddetermine the above characteristics of porosity, pore size, thicknessand particle diameter. Additionally, the substrate can be made of analkoxysilane, or colloidal silicon dioxide or both in varyingconcentrations. It is also known in the art that some tolerance fortrace impurities is allowed. The alkoxysilanes include trialkoxysilanesand tetraalkoxysilanes.

[0085] Functionalization/Silylation

[0086] The porous silica substrate of the present invention can besilylated to provide many functionalized attachments. Alternatively, thecolloidal silica particles, which are used in preparing the poroussilica substrate, can be functionalized so that the porous silicasubstrate that is formed is already functionalized. The silylation canbe accomplished by using any number of silylating agents. Manysilylating agents are known in the art. For example,N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide (PCR Inc.,Gainesville, Fla.) has been used to silylate a glass substrate prior tophotochemical synthesis of arrays of polynucleotides on the substrate,as described in McGall et al., J. Am. Chem. Soc., 119:5081-5090 (1997),the disclosure of which is incorporated herein by reference.

[0087] Hydroxyalkylsilyl compounds that have been used to preparehydroxyalkylated substances, such as glass substrates.N,N-bis(hydroxyethyl) aminopropyltriethoxysilane has been used to treatglass substrates to permit the synthesis of high-density polynucleotidearrays. See McGall et al., Proc. Natl. Acad. Sci., 93:13555-13560(1996); and Pease et al., Proc. Natl. Acad. Sci., 91:5022-5026 (1994),the disclosures of which are incorporated herein.Acetoxypropyl-triethoxysilane and 3-Glycidoxy propyltrimethoxysilanehave been used to treat glass substrates to provide a linker for thesynthesis of polynucleotides. See EP Patent Application No. 89 120696.3.

[0088] The functionalized silicon compounds include an activated silicongroup and a derivatizable functional group. Exemplary derivatizablefunctional groups include hydroxyl, amino, carboxyl and thiol, as wellas modified forms thereof, such as activated or protected forms. Thefunctionalized silicon compounds may be covalently attached to surfacesto form functionalized surfaces that may be used in a wide range ofdifferent applications. The silicon compounds are attached to thesurface of a substrate comprising silica, such as a glass substrate, toprovide a functionalized surface on the silica containing substrate, towhich molecules, including polypeptides and nucleic acids, may beattached. After covalent attachment of a functionalized silicon compoundto the surface of a solid silica substrate to form a functionalizedcoating on the substrate, an array of nucleic acids may be covalentlyattached to the substrate or synthesized off of the functional groups.Thus, the method permits the formation of high density arrays of nucleicacids immobilized on a substrate, which may be used in conducting highvolume nucleic acid hybridization assays.

[0089] As used herein, the term “silicon compound” refers to a compoundcomprising a silicon atom. In a preferred embodiment, the siliconcompound is a silylating agent comprising an activated silicon group,wherein the activated silicon group comprises a silicon atom covalentlylinked to at least one reactive group, such as an alkoxy or halide, suchthat the silicon group is capable of reacting with a functional group,for example on a surface of a substrate, to form a covalent bond withthe surface. Exemplary activated silicon groups include —Si(OMe)₃;—SiMe(OMe)₂; —SiMeCl₂; SiMe(OEt)₂; SiCl₃ and —Si(OEt)₃.

[0090] As used herein, the term “functionalized silicon compound” refersto a silicon compound comprising a silicon atom and a derivatizablefunctional group. In a preferred embodiment, the functionalized siliconcompound has an activated silicon group and a derivatizable functionalgroup. “Derivatizable functional group” refers to a functional groupthat is capable of reacting to permit the formation of a covalent bondbetween the silicon compound and another substance, such as a polymer ora polymer building block. Exemplary derivatizable functional groupsinclude hydroxyl, amino, carboxyl and thiol, as well as modified formsthereof, such as activated or protected forms. Derivatizable functionalgroups also include substitutable leaving groups such as halo orsulfonate. In one preferred embodiment, the derivatizable functionalgroup is a group, such as a hydroxyl group, that is capable of reactingwith activated nucleotides to permit nucleic acid synthesis.

[0091] The surface can be functionalized by covalently attaching to thesurface a functionalized silicon compound, wherein the functionalizedsilicon compound comprises at least one derivatizable functional groupand a plurality of activated silicon groups, for example, 2, 3, 4 ormore activated silicon groups. An array of nucleic acids can becovalently attached to the functionalized silicon compounds on thesurface. The number of silicon groups and the number of derivatizablefunctional groups in the silicon compound may be modified for differentapplications, to increase or decrease the number of bonds to a supportsuch as a glass support.

[0092] Further description of several silylating agents and methods fortheir preparation can be found in U.S. Pat. No. 5,624,711 and in U.S.Ser. No. 09/172,190, filed Oct. 13, 1998, which are hereby incorporatedby reference. Commercially available silicon compounds and a review ofsilicon compounds is provided in Arkles, Ed., “Silicon, Germanium, Tinand Lead Compounds, Metal Alkoxides, Diketonates and Carboxylates, ASurvey of Properties and Chemistry,” Gelest, Inc., Tullytown, Pa.(1995), the disclosure of which is incorporated herein. Functionalizedsilicon compounds may be synthesized using methods available in the artof organic chemistry, for example, as described in March, AdvancedOrganic Chemistry, John Wiley & Sons, New York (1985).

[0093] Polymer Coated Porous Substrate

[0094] The porous substrate of the present invention can bepolymer-coated. The substrate can be polymer-coated using dip coating,covalent polymer attachment, in situ polymerization, or combinationsthereof. In yet another aspect, the substrate can be glycan-coated.While similar to the polymer-coated supports, the properties ofglycan-coated supports can be quite different and provide extremelyhydrophilic surfaces that are useful in binding assays and diagnosticapplications. A detailed description of polymer and glycan coatingmaterials and methods is given in the U.S. Pat. No. 5,624,711, which ishereby incorporated by reference.

[0095] In any of these methods, the choice of available surface polymersis extensive. Suitable polymers include chloromethylatedstyrene-divinylbenzene (Merrifield resin), phenylacetamidomethylatedstyrene-divinylbenzene (PAM resin), and crosslinked polyethyleneglycol-polystyrene grafts (TentaGel resin). The polymers which are usedto coat the solid support can also be selected based upon theirfunctional groups which will serve as synthesis initiation sites.Typically, polymers having primary amine, carboxyl or hydroxylfunctional groups will be selected.

[0096] Polymers having primary amine functional groups are of interestas these polymers can be readily adapted to coupling chemistry currentlyused in the high density array synthesis. Suitable polymers havingprimary amine functional groups include polyethyleneimine linear orbranched polymers, polyacrylamide, and polyallylamine which are allcommercially available. Other polymers, such as polydimethylacrylamide(or other polymers in this genus), can be synthesized according topublished procedures (see Atherton, E. et al. in “Solid Phase PeptideSynthesis: A Practical Approach,” Chapter 4, pp. 39-45, IRL Press(1989); and Arshady, R. et al., J. Chem. Soc. Perkin. Trans. 1:529(1981)).

[0097] Polymers having carboxyl functional groups are also useful as theresulting surfaces are very hydrophilic. Furthermore, the synthesisinitiation sites (i.e. the carboxylic acid groups) are useful in peptidesynthesis which proceeds from the amino terminus of the peptide to thecarboxylic acid terminus. Suitable polymers having carboxylic acidfunctional groups include poly(acrylic acid), poly(ethylene/maleicanhydride), and poly(methylvinyl ether/maleic anhydride).

[0098] Polymers having hydroxyl functional groups are also useful as theresulting surfaces are extremely wettable. Examples of suitable polymersinclude polyethyleneglycol (PEG), polyvinyl alcohol and carbohydrates.

[0099] In general, the glycan-coated surfaces can be prepared in amanner analogous to the preparation of polymer-coated surfaces usingcovalent attachment. Thus, a glass surface can be modified (silanized)with reagents such as aminopropyltriethoxysilane to provide a glasssurface having attached functional groups (in this case, aminopropylgroups). The modified surface is then treated with a solution of amodified dextran to provide a surface having a layer of dextran which iscovalently attached.

[0100] Linking Molecules

[0101] After derivatization of the porous substrate, the derivatizedsurface may be contacted with a mixture of linking molecules and diluentmolecules (the diluent molecules are optional and are not included inpreferred embodiments). The diluent molecules for example have only onecenter which is reactive with the reactive sites on the derivatizedsubstrate surface. All the other reactive centers on the diluentmolecules are protected, capped or otherwise rendered inert. The linkingmolecules will similarly have one center which is reactive with thereactive sites on the derivatized substrate surface. Additionally, thelinking molecules will have a functional group which is optionallyprotected and which can later serve as a synthesis initiation site. Thelinking and diluent molecules are present in the mixture in a ratiowhich is selected to control the functional site density on the surface.The ratio of linking molecules to diluent molecules is for example fromabout 1:2 to about 1:200, e.g., from about 1:10 to about 1:50.Alternatively, the ratio of linking molecules to diluent molecules canbe from about 200:1, or from about 100:1, or from about 10:1, as desiredfor adjusting the density of polymers on the surface.

[0102] The linking molecules should be of sufficient length to permitpolymers synthesized thereon to interact freely with molecules exposedto the polymers. The linking molecules should be 3-50 atoms long toprovide sufficient exposure of ligands to their receptors. Typically,the linking molecules will be aryl acetylene, ethylene glycol oligomerscontaining 2-14 monomer units, diamines, diacids, amino acids, peptides,or combinations thereof. In some embodiments, the linking molecule canbe a nucleotide or a polynucleotide. The particular linking moleculeused can be selected based upon its hydrophilic/hydrophobic propertiesto improve presentation of the polymer synthesized thereon to certainreceptors, proteins or drugs.

[0103] The linking molecules can be attached to the substrate bysiloxane bonds (using, for example, glass or silicon oxide surfaces).Siloxane bonds with the surface of the substrate may be formed in oneembodiment via reactions of linking molecules bearing traditionalaminopropyl silane groups such as trichlorosilyl, trimethoxy ortriethoxy silyl groups. The linking molecules may optionally be attachedin an ordered array, i.e., as parts of the head groups. In some aspects,the linking molecules are absorbed to the surface of the substrate. Inaddition, linking molecules may also be present in case of nucleic acidsynthesis and hybridization assays.

[0104] As noted above, the linking molecule, prior to attachment to thederivatized surface has an appropriate functional group at each end, onegroup appropriate for attachment to the reactive sites on a derivatizedsurface and the other group appropriate as a synthesis initiation site.For example, groups appropriate for attachment to the derivatizedsurface would include amino, hydroxy, thiol, carboxylic acid, ester,amide, isocyanate and isothiocyanate. Additionally, for subsequent usein synthesis of polymer arrays or libraries, the linking molecules usedherein will typically have a protecting group attached to the functionalgroup on the distal or terminal end of the linking molecule (oppositethe solid support).

[0105] The linking molecule contributes to the net hydrophobic orhydrophilic nature of the surface. For example, when the linkingmolecules comprise a hydrocarbon chain, such as ——(CH₂)_(n)——, theeffect is to decrease wettability. Linking molecules likepolyoxyethylene (——(CH₂CH₂O_(n))—, or polyamide (——(CH₂CONH)_(n)——)chains tend to make the surface more hydrophilic (i.e., increasewettability).

[0106] The diluent molecules can be any of a variety of molecules whichcan react with the reactive sites present on the derivatized substrateand which generally have remaining functional groups capped orprotected. The diluent molecules can also be selected to imparthydrophobic or hydrophilic properties to the substrate surface. Forexample, the diluent molecules are alkanoic acids, which imparthydrophobic properties to the surface. In other cases, the diluentmolecules are amino acids, wherein the amine and any side chainfunctionality which is present are protected. In these instances, thediluent molecules can contain functionality which is altered upontreatment with various reagents such as acid, base or light, to generatea surface having other desired properties. For example, use of O-t-Butylserine as a diluent molecule provides a hydrophobic surface duringpolymer synthesis, but upon treatment with acid (cleaving the t-butylether), a more hydrophilic surface is produced for assays.

[0107] Thus, after reacting the mixture of linking molecules and diluentmolecules with the surface and subsequently synthesizing a desiredpolymer onto the functional sites on the linking group, the protectinggroups on the surface-attached diluent molecules are removed to providea more hydrophilic (i.e. “wettable”) surface. In preferred embodiments,the diluent molecules are protected glycine, protected serine, glutamicacid or protected lysine. Dimethyl N,N-diiosopropylphosphoramidite canbe used to phosphorylate surface hydroxyls, to alter the surfacecharacteristics. Further description of linking molecules and diluentmolecules are given in the U.S. Pat. No. 5,624,711, which is herebyincorporated by reference.

[0108] Substrates with Acidic Surfaces

[0109] The present invention also provides porous substrates which arederivatized to provide acidic surfaces, or “carboxy chips.” The carboxychips can be considered as “reverse polarity” surfaces (as compared withthe more typical aminopropylsilane derivatized surfaces). Such reversepolarity surfaces will find application in combinatorial synthesisstrategies which require a carboxylic acid initiation site. For example,peptide synthesis which is carried out from the N-terminal end to theC-terminal end can be carried out on a carboxy chip. Additionally, smallmolecules such as prostaglandins, β-turn mimetics and benzodiazepinescan also be synthesized on a carboxy chip. Carboxy chips will also findapplication in the preparation of chips having synthesis initiationsites which are amines. In this aspect, the carboxy chips will bereacted with a suitably protected alkylenediamine to generate an aminosurface.

[0110] Carboxy chips can be prepared by a variety of methods. Forexample, a solid support is derivatized with an aminoalkylsilane toprovide a surface of attached amino groups. The derivatized surface isthen treated with an anhydride such as glutaric anhydride to acylate theamino group and provide a surface of carboxylic acid functionalities.Alternatively, the aminoalkylsilane is first reacted with an anhydride(i.e., glutaric anhydride) to generate a carboxylic acid silane whichcan then be coupled to the porous substrate, and similarly provide asurface of carboxylic acid residues. Further description of carboxychips can be found in the U.S. Pat. No. 5,624,711, which is herebyincorporated by reference.

[0111] Array Synthesis

[0112] Large scale chemical diversity on primarily inorganic poroussubstrates can be achieved by synthetic strategies and devices presentedherein. The preferred substrates, solid-phase chemistry, photolabileprotecting groups, deprotection techniques, and photolithography, whenbrought together, achieve very high density, spatially-addressable,parallel chemical synthesis. Thus, the preferred substrates providedherein can be used in a number of applications, including light-directedmethods, flow channel and spotting methods, pin-based methods andbead-based methods (see the patents and references cited above).

[0113] Alternatively, the primarily inorganic porous substrates of thepresent invention can be used to prepare high density arrays of polymersusing conventional linkage chemistry-based synthetic methods, also knownas phosphoramidite-based synthesis methods. One example of conventionallinkage chemistry-based polynucleotide synthesis is known as standarddimethoxytrityl (DMT) method. Examples of this and additionalphosphoramidite synthesis methods are described in the “User Manual forApplied Biosystems Model 391,” pp. 6-1 to 6-24, available from AppliedBiosystems, 850 Lincoln Center Dr., Foster City, Calif. 94404, and aregenerally known by those skilled in the art. See also M. Gait,Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press,London.

[0114] Light-Directed Methods

[0115] “Light-directed” methods (which are one technique in a family ofmethods known as VLSIPS™ methods) are described in U.S. Pat. No.5,143,854, '305, '934 and other patents above all of which areincorporated by reference. The light directed methods discussed in thesepatents involve activating known locations or predefined regions of asubstrate or solid support and then contacting the substrate with apreselected solution of monomers or polymers. The known locations orpredefined regions can be activated with a light source, typically shownthrough a mask (much in the manner of photolithography techniques usedin integrated circuit fabrication). Other regions of the substrateremain inactive because they are blocked by the mask from illuminationand remain chemically protected. Thus, a light pattern defines whichregions of the substrate react with a given monomer. By repeatedlyactivating different sets of predefined regions and contacting differentmonomer solutions with the substrate, a diverse array of polymers isproduced on the substrate. Of course, other steps such as washingunreacted monomer solution from the substrate can be used as necessary.

[0116] The porous silica substrate and the optionally provided linkermolecules thereon can be the same as described infra in the context ofconventional linkage chemistry-based synthesis. The linker molecules mayeach include a protecting group. In light-directed polymer synthesis,the protecting group is a photocleavable (photoreactive) protectinggroup. Photocleavable protecting groups, addition, binary synthesisstrategy, and other processes associated with light directed methods areshown in U.S. Pat. No. 5,744,305.

[0117] Flow Channel or Spotting Methods

[0118] Additional methods applicable to library synthesis on a singlesubstrate are described in U.S. Pat. Nos. 5,677,195, 5,384,261, and6,040,138 incorporated herein by reference for all purposes. In themethods disclosed in these applications, reagents are delivered to thesubstrate by either (1) flowing within a channel defined on predefinedregions or (2) “spotting” on predefined regions. However, otherapproaches, as well as combinations of spotting and flowing, may beemployed. In each instance, certain activated regions of the substrateare mechanically separated from other regions when the monomer solutionsare delivered to the various reaction sites. One of ordinary skill inthe art would also appreciate that this method can also be used todeposit pre-synthesized oligomers or polymers for furtherpolymerization.

[0119] The “spotting” methods of preparing arrays of the presentinvention can be implemented in much the same manner as the flow channelmethods. For example, a monomer A can be delivered to and coupled with afirst group of reaction regions which have been appropriately activated.Thereafter, a monomer B can be delivered to and reacted with a secondgroup of activated reaction regions. Unlike the flow channel embodimentsdescribed above, reactants are delivered by directly depositing (ratherthan flowing) relatively small quantities of them in selected regions.In some steps, of course, the entire substrate surface can be sprayed orotherwise coated with a solution. In preferred embodiments, a dispensermoves from region to region, depositing only as much monomer asnecessary at each stop. Typical dispensers include a micropipette, aquill, or a pin and ring to deliver the polymer solution to thesubstrate and a robotic system to control the position of themicropipette with respect to the substrate, or an ink-jet printer. Inother embodiments, the dispenser includes a series of tubes, a manifold,an array of pipettes, or the like so that various reagents can bedelivered to the reaction regions simultaneously.

[0120] Pin-Based Methods

[0121] Another method which is useful for the preparation of compoundsand libraries of the present invention involves “pin based synthesis.”This method is described in detail in U.S. Pat. No. 5,288,514,previously incorporated herein by reference. The method utilizes asubstrate having a plurality of pins or other extensions. The pins areeach inserted simultaneously into individual reagent containers in atray. In a common embodiment, an array of 96 pins/containers isutilized.

[0122] Each tray is filled with a particular reagent for coupling in aparticular chemical reaction on an individual pin. Accordingly, thetrays will often contain different reagents. Since the chemistrydisclosed herein has been established such that a relatively similar setof reaction conditions may be utilized to perform each of the reactions,it becomes possible to conduct multiple chemical coupling stepssimultaneously. In the first step of the process the invention providesfor the use of substrate(s) on which the chemical coupling steps areconducted. The substrate is optionally provided with a spacer havingactive sites. In the particular case of polynucleotides, for example,the spacer may be selected from a wide variety of molecules which can beused in organic environments associated with synthesis as well asaqueous environments associated with binding studies. Examples ofsuitable spacers are polyethyleneglycols, dicarboxylic acids, polyaminesand alkylenes, substituted with, for example, methoxy and ethoxy groups.

[0123] Additionally, the spacers will have an active site on the distalend. The active sites are optionally protected initially by protectinggroups. Among a wide variety of protecting groups which are useful areFMOC, BOC, t-butyl esters, t-butyl ethers, and the like. Variousexemplary protecting groups are described in, for example, Atherton etal., “Solid Phase Peptide Synthesis,” IRL Press (1989), incorporatedherein by reference. In some embodiments, the spacer may provide for acleavable function by way of, for example, exposure to acid or base.

[0124] Bead Based Methods

[0125] A general approach for bead based synthesis is described in U.S.Pat. Nos. 5,770,358, 5,639,603, and 5,541,061 the disclosures of whichare incorporated herein by reference. For the synthesis of moleculessuch as polynucleotides on beads, a large 5 plurality of beads aresuspended in a suitable carrier (such as water) in a container. Thebeads are provided with optional spacer molecules having an active site.The active site is protected by an optional protecting group.

[0126] In a preferred embodiment, the beads are tagged with anidentifying tag which is unique to the particular double-strandedpolynucleotide or probe which is present on each bead. A completedescription of identifier tags for use in synthetic libraries isprovided in U.S. Pat. No. 5,639,603.

[0127] Conventional Linkage Chemistry Methods

[0128] In addition to the above-described light-directed methodology,high density arrays of polymers can be synthesized using conventionallinkage-based chemistry and using protecting groups that are chemicallycleaved using solution or vapor-phase deprotection agents. Thismethodology is described in greater detail in the U.S. Pat. No.5,599,695, which is incorporated by reference.

[0129] The above methodology can be applied to the synthesis of severaltypes of polymer, including those of biological interest such aspolynucleotides, nucleic acids, polypeptides, proteins, oligosaccharidesand polysaccharides. Chemical synthesis of polypeptides is known in theart and are described further in Merrifield, J., J. Am. Chem. Soc., 91:501 (1969); Chaiken I. M., CRC Crit. Rev. Biochem., 11: 255 (1981);Kaiser et al., Science, 243:187 (1989); Merrifield, B., Science, 232:342(1986); Kent, Ann. Rev. Biochem., 57: 957 (1988); and Offord, R. E.,Semisynthetic Proteins, Wiley Publishing (1980), which are incorporatedherein by reference). In addition, methods for chemical synthesis ofpeptide, polycarbamate, and polynucleotide arrays have been reported(see Fodor et al., Science, 251:767-773 (1991); Cho et al., Science,261:1303-1305 (1993), each of which is incorporated herein byreference).

[0130] Data Collection

[0131] Devices to detect regions of a substrate which containfluorescent markers are known in the art. See e.g., U.S. Pat. Nos.5,631,734; 5,744,305; 5,981,956 and 6,025,601, incorporated byreference. These devices would be used, for example, to detect thepresence or absence of a labeled receptor such as an antibody which hasbound to a synthesized polymer on a substrate.

[0132] U.S. Pat. No. 5,527,681, the disclosure of which is incorporatedherein, describes use of computer tools for forming arrays. For example,a computer system may be used to select nucleic acid or other polymerprobes on the substrate, and design the layout of the array as describedin U.S. Pat. No. 5,571,639, the disclosure of which is incorporatedherein.

[0133] Further understanding of the nature and advantages of theinventions herein may be realized by reference to the remaining portionsof the specification and the attached drawings.

[0134] All publications cited herein are incorporated herein byreference in their entirety.

APPLICATIONS

[0135] The above-described arrays of polymers such as polypeptides, ornucleic acids or polysaccharides prepared on the porous substrates ofthis invention can be used in a variety of applications includingbiological binding assays and nucleic acid hybridization assays. Forexample, polynucleotide or nucleic acid arrays can be used to detectspecific nucleic acid sequences in a target nucleic acid. See, e.g., PCTpatent publication Nos. WO 89/10977 and 89/11548. General hybridizationand detection of nucleic acids is shown in U.S. Pat. Nos. 5,631,734,5,510,270 and 5,324,633.

[0136] Methods for screening target molecules for specific binding toarrays of polymers, such as nucleic acids, immobilized on a solidsubstrate, are disclosed, for example, in U.S. Pat. Nos. 5,677,195,5,631,734, 5,624,711, 5,599,695, 5,510,270, 5,445,934, 5,451,683,5,424,186, 5,412,087, 5,405,783, 5,384,261, 5,252,743 and 5,143,854;5,800,992, 5,795,716, 6,040,138 and U.S. application Ser. No.08/388,321, filed Feb. 14, 1995. Accessing genetic information usinghigh density DNA arrays is further described in Chee, Science274:610-614 (1996). Arrays to detect mutations in reference. Arrays todetect mutations in HIV genes are described in detail in the U.S. Pat.No. 5,861,242. Arrays to detect nucleic acid from nonviral pathogensinfecting AIDS patients are shown in U.S. Pat. No. 5,861,242. Devicesfor concurrently processing multiple biological chip assays may be usedas described in U.S. Pat. No. 5,545,531. The arrays of polynucleotidesor nucleic acids prepared on porous silica substrates provided hereincan be used to screen polymorphisms in samples of genomic material. Thedetailed methodology is provided in the U.S. Pat. No. 5,858,659. Tilingstrategies are discussed in detail in the U.S. Pat. No. 5,837,832.Hybridization and scanning are generally carried out by methodsdescribed in, e.g., Published PCT Application Nos. WO 92/10092 and WO95/11995, and U.S. Pat. No. 5,424,186, incorporated herein by reference.Gene expression may be monitored by hybridization of large numbers ofmRNAs in parallel using high density arrays of nucleic acids in cells,such as in microorganisms such as yeast, as described in Lockhart etal., Nature Biotechnology, 14:1675-1680 (1996), and U.S. Pat. Nos.5,800,992 and 6,040,138, PCT WO 97/10365. Bacterial transcript imagingby hybridization of total RNA to nucleic acid arrays may be conducted asdescribed in Saizieu et al., Nature Biotechnology, 16:45-48 (1998), thedisclosure of which is incorporated herein. Additional examples ofapplications in biomedical and genetic research and clinical diagnosticsare disclosed in U.S. Pat. Nos. 5,547,839, 5,710,000 (using Type-IIsrestriction endonucleases), and in U.S. patent application Ser. No.08/143,312. Other applications include chip based genotyping, speciesidentification and phenotypic characterization, as described in U.S.patent application Ser. No. 08/797,812, filed Feb. 7, 1997, and U.S.application Ser. No. 08/629,031, filed Apr. 8, 1996.

[0137] Arrays of nucleic acids for use in gene expression monitoring aredescribed in PCT WO 97/10365, the disclosure of which is incorporatedherein. In one embodiment, arrays of nucleic acid probes or otherpolymer probes are immobilized on a surface, wherein the array comprisesmore than 100 different nucleic acids and wherein each different nucleicacid is localized in a predetermined area of the surface, and thedensity of the different nucleic acids is greater than about 60different nucleic acids per 1 cm².

[0138] Arrays of nucleic acids or other polymers immobilized on asurface also are described in detail in U.S. Pat. No. 5,744,305, thedisclosure of which is incorporated herein. As disclosed therein, on asubstrate, nucleic acids or other polymers with different sequences canbe immobilized each in a predefined area on a surface. For example, 10,50, 60, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ different monomer sequencesmay be provided on the substrate. The nucleic acids of a particularsequence are provided within a predefined region of a substrate, havinga surface area, for example, of about 1 cm² to 10⁻¹⁰ cm². In someembodiments, the regions have areas of less than about 10⁻¹, 10⁻², 10⁻³,10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, or 10⁻¹⁰ cm². For example, in oneembodiment, there is provided a planar, non-porous support having atleast a first surface, and a plurality of different nucleic acidsattached to the first surface at a density exceeding about 400 differentnucleic acids/cm², wherein each of the different nucleic acids isattached to the surface of the solid support in a different predefinedregion, has a different determinable sequence, and is, for example, atleast 4 nucleotides in length. The nucleic acids may be, for example,about 4 to 20 nucleotides in length. The number of different nucleicacids may be, for example, 1000 or more.

[0139] Since the application of a porous glass is considerably broaderthan its use in the examples discussed and provided herein, theseexamples are used for illustrative purposes only and should not beconstrued as a limitation on the full scope of the invention disclosedherein.

EXAMPLES Example 1

[0140] Sol-Gel Silica Deposition

[0141] Methods and Materials

[0142] Glass microscope slides (2 in×3 in×0.027 in, from ERIESCIENTIFIC) were cleaned in piranha solution (30% v/v hydrogen peroxide,70% v/v sulfuric acid) for 30 minutes with gentle stirring. They werethen transferred immediately to clean water in which they were stored.The slides were removed and blown dry with dry nitrogen immediatelyprior to film deposition. Long-term storage in water is not necessaryfor The starting point for the film preparation is to mix the precursorsin solution. Colloidal silica, tetramethoxysilane (TMOS), hydrochloricacid, water, and methanol are mixed in solution. The particle size ofthe colloidal silica ranged from about 12 nm to >100 nm and wasdeposited on a glass surface. In addition to the glass slides discussedabove, other exemplary glass providing a surface for the deposition ofcolloidal silica is soda lime glass (Erie Scientific), as well asborofloat glass or fused silica glass (U.S. Precision Glass, Elgin,Ill.). The resultant pores from such particles are in the 2-40 nm range.Layers having a thickness of about 0.1 μm-2 μm were investigated, butthe thickness was chosen for experimental purposes and actual devicesmay have thicker or thinner layers. TMOS may be added to the colloidalsilica to strengthen the resulting silica matrix.

[0143] Two sources of colloidal silica were used. LUDOX™ was purchasedfrom E. I. Dupont de Nemoirs, and includes silica spheres suspended inwater, stabilized by sodium counterions. For HS-40, the spheres arenominally 12nm diameter, whereas for TM-40 they are 22 nm. SNOWTEX™colloidal silica was purchased in from NISSAN CHEMICALS in three sizes(listed size range in parenthesis): SNOWTEX-50 (20-30 nm), SNOWTEX-20L(40-50 nm), and SNOWTEX-ZL (70-100 nm).

[0144] Tetramethoxysilane was purchased from ALDRICH, with specificationof 98% purity. The pH of the total mixture was adjusted to 3.75+/−0.25,measured with a VWR benchtop pH meter Model 8015™ (VWR SCIENTIFIC).

[0145] Formation of Films

[0146] Equal weights of colloidal silica were used in each solution, andthen water was added as necessary to keep the solution volume constant.Table 1 shows the compositions that were used. 1.2M HCl was used totitrate the solutions to the appropriate pH, with different amountsbeing required in each case. It should be noted that the glasscomposition listed in the Table below may also have trace amounts ofTABLE 1 Representative Colloidal Silica Solution Compositions (amountsin mL) Size (nominal) 12 nm 2 nm 20-30 nm 40-50 nm 70-100 nm Brand(Ludox HS-40) (Ludox TM-40) (Snowtex-50) (Snowtex-20L) (Snowtex-ZL)Water 13.4 13.4 14.9 5.2 13.4 Methanol 12.6 12.6 12.6 12.6 12.6Colloidal Silica 6.6 6. 5.2 14.8 6.6 TMOS 1.36 1.36 1.36 1.36 1.36

[0147] The components were mixed in solution and stirred gently forapproximately 10 minutes. Stirring was discontinued, and the mixture wasleft to react for approximately one hour. The solution was then filteredwith a 0.45 micron filter (either with a filter attached to the syringeor by filtering the solution separately and then dispensing). Films werealso created without the use of TMOS in the precursors. For those films,using the SNOWTEX-20L and ZL, the solution was typically diluted to10-20 wt. % with pure water, and then filtered with a syringe filter (orHPLC-grade nylon 0.45 u filter paper for large batches). These caseswill be noted in later sections. Prior to deposition, dynamic lightscattering was performed on the solutions. Light scattering analysisgives the size distribution of the particles in solution.

[0148] The substrate was then spun at various speeds, ranging from 500to 2500 rpm on a Headway Research photo-resist spin coater (HeadwayResearch) to distribute the solution into a thin film. For the datareported, the films were deposited in a lab environment with no explicitcontrol of air flow, temperature, or humidity.

[0149] The substrates were then allowed to dry at room temperatureconditions for several hours. They were then transferred to an oven werethey were fired at 75° C. for one hour, 100° C. for 2 hours, and then500° C. for 5 hours. The first two steps are to remove residualmoisture, the last to cause mild sintering of the matrix for mechanicalstability. Some films were also created without the use of thisprocedure, and will be noted in later sections. In a preferredembodiment, no sintering (annealing) step is performed. Such a porouslayer also has improved effective surface area because sintering orsimilar high-temperature processing generally causes particles to atleast partially combine or densify. It has been found that theun-sintered colloidal silica layers are sufficiently robust for avariety of intended uses, such as polymer synthesis or bioassay.

[0150] Thickness of the films was measured by scratching the films andthen measuring the depth of scratch with a surface profilometer, such asa DEKTAK II™ available from VEECO. Surfaces for ellipsometricmeasurements were made by depositing the films on silicon rather thanglass to provide a contrast in the index of refraction. The silicondioxide surface layer on the silicon substrate should provide asubstrate similar to the glass surface. Ellipsometric measurements weremade with a Gaertner Ellipsometer Model L116C™ with a light wavelengthof 632.8 nm (Gaertner Scientific Corporation).

[0151] A desirable feature of the porous layers of the present inventionformed on the surface of the underlying substrate is that they do notappreciably scatter light, such as may be used in scanning arrays ofpolymer or genetic materials. In some prior art processes, relativelylarge holes have been etched into the surface of a substrate, such as aglass substrate. Such large holes are distinguishable from the pores ofthe present invention in that a process mask or similar method istypically used to define the location of the holes. Furthermore, suchholes can be large enough to scatter light and interfere with scanningor other light-based use of the substrate.

[0152] After the porous surfaces were prepared they needed to besilanated to appropriately prepare the surface for oligomer synthesis.Prior to silanation, the dried and annealed slides were soaked in waterfor approximately 15 minutes. They were then transferred to a bath ofreagent alcohol for 3 minutes, and then to the silane bath for 15minutes. The silane bath consists of 94% reagent alcohol, 5% water, and1% bis (2 hydroxyethyl)-3-aminopropyltriethoxysilane in 62% ethanol(Gelest Inc.) by volume. Slides were then transferred to two successiveisopropanol baths (5 minutes each), blown dry with dry nitrogen, andplaced in a 35-50° C. oven for 3 minutes. The above silanationconditions were used for most applications. However other silanes andsilylation conditions were also used.

[0153] Fluoreprime Stain Assay: Qualitative/Quantitative SurfacePerformance using Photochemical Polynucleotide Synthesis

[0154] Quantitative studies of the synthesis, density and uniformity ofthe porous silica substrates was conducted using methods based onsurface fluorescence as described in McGall et al; J. Am. Chem. Soc.;119: 5081-5090 (1997). Fluorescent “staining” of the surface wasperformed as described, with the exception that a fluoresceinconcentration of 0.5 mM in a solution containing 50 mM DMT-T-CEP inacetonitrile was used. The fluorescein phosphoramidite is coupled to thefree hydroxyl groups with the standard protocol. Substrates are thendeprotected for a minimum of one hour in a 1:1 solution ofethylenediamine/ethanol, rinsed with deionized water, and blown dry withdry nitrogen. The substrate is then scanned. using a confocalmicroscopy. The signal obtained is a function of the number of availablehydroxyl groups on the surface. In this case, the relative values ascompared to other types of similarly treated glass is an indication ofthe relative density and capacity of the surface. This technique alsoprovides a visual picture of the surface with respect to quality andunformity of the surface. The technique is not limited to hydroxylgroups but may be modified to measure other groups of interest forsupport of polymer of interest on the surface by using the appropriatelyfunctionalized molecule for detection.

[0155] HPLC Quantitation Assay

[0156] This technique is described in U.S. Pat. No. 5,843,655, “Methodsfor Testing Oligonucleotide Arrays,” also by Affymetrix. HPLC (highperformance liquid chromatography) analyses were performed on a BeckmanSystem Gold ion exchange column using fluorescence detection at 520 nm.Elution is performed with a linear gradient of 0.4M NaClO₄ in 20 mM TrispH 8, at a flow rate of 1 mL/min, or other suitable buffer system.

[0157] The HPLC quantitation assay was used to measure the site densityavailable for generating polymers, the coupling efficiency of eachsubsequent addition of monomer to the growing chain, and the extent ofadsorption or entrapment of the reagents within the porous matrix.

[0158] In this technique, MeNPOC PEG is attached to the surface,followed by a capping step, a cleavable (SO₂) linker, a spacer molecule(“C3,” a three carbon chain), and a fluorophore. The linker is5′-phosphate-ON reagent (ChemGenes Corporation), and the fluorophore is5-carboxyfluorescein-CX CED phosphoramidite (Flam) (BioGenex). Thepurpose of the spacer molecule is to discriminate between fluorescentmolecules that have attached to the intended synthesis sites vs. thosethat have remained on the surface without chemical attachment. It shouldbe noted that the cleavable linker can be attached directly to the glasssurface without the use of a PEG- type linker. Synthesis was alsoaccomplished on the surfaces using traditional acid-based polynucleotidechemistry (trityl chemistry). Similar chemistries can be applied for thesynthesis of polynucleotide, peptide, oligosaccarides, peptide nucleicacids, and other polymers. The description relating to the peptidenucleic acids can be found in the PCT publication WO92/20702, publishedNov. 26, 1992, which is incorporated by reference in its entirety.

[0159] After synthesis the surface is treated with a known solutionvolume of reagent necessary to cleave the linker to release3′-C₃-flam-5′, and this is typically cleaved in solution overnight (1:1by volume ethylenediamine/water). The resulting solution is diluted andcoinjected with an internal standard onto and analyzed by HPLC. Theinternal standard is a 3′-C₃-C₃-Flam-5′ chain prepared separately on anABI synthesizer. Concentration is determined by UV-Vis spectra on aVarian Cary 3E spectrophotometer (Varian). Integration of HPLC peakareas can be used to determine total site density and cleanliness ofcoupling.

[0160] In an analogous experiment step by step coupling yields can bedetermined by coupling the cleavable linker amidite and Fluoresceinamidite to the surface followed by synthesis of a polymer of interestusing MeNPOC chemistry. Typically a 6-mer homopolymer such as poly (A)₆is synthesized. The probes are then cleaved in solution, diluted with anappropriate corresponding internal standard, and run through the HPLCcolumn. Peaks are observed for the probes of lengths 1-6, and giveindication of the relative coupling yield of successively added bases.

[0161] Synthesis of Full-length Probes for Functional Assessment ofPorous Surface under Hybridization Conditions

[0162] Full length probes capable of hybridization, typically 20-merprobes, were synthesized using Affymetrix synthesizers as described inU.S. Pat. No. 5,405,783, using nucleoside phosphoramidites equipped with5′- photolabile MeNPOC protecting groups. The sequence used for themajority of the preliminary experiments is (3′)-AGG TCT TCT GGT CTC CTTTA (5′), with the 3′ end attached to the surface. The non-photolabileprotecting groups were removed post synthesis in 1:1ethylenediamine/ethanol (v/v) for a minimum of 4 hours.

[0163] Hybridization assays were performed on the 2×3″ slides withoutfurther processing. Each slide was placed in 10-15 mls of 10-50 nMtarget oligonucleotide in hybridization buffer with gentle stirring. Thetwo hybridization buffers commonly used are 6×SSPE and MES (sodiumchloride, sodium phosphate, EDTA and 2-[N-morpholino]ethanesulfonic acidrespectively). The target sequence is the exact complement of the probesequence: (5′) Fluorophore-TCC AGA AGA CCA GAG GAA AT.

[0164] The pattern and intensity of surface fluorescence was imaged witha specially constructed scanning laser confocal fluorescence microscope,as described in McGall, supra. Where necessary, the photon multipliertube gain was adjusted to keep signals within range for the detector.

[0165] Results

[0166] Initial testing of the porous films was conducted with thesmaller particle sizes (Ludox) and including the TMOS and annealingsteps. Deposition of the particles on the surface of the substrate hasbeen confirmed by using scanning electron microscopy and atomic forcemicroscopy. For films deposited at 2500, 1500, and 500 rpm with theTM-40 solution, the ratio of the hybridization signal on porous glass toflat glass reaches factors of approximately 5, 7 and 18 times that offlat glass respectively. The ratios are calculated by dividing thehybridization signal from the porous surface (with backgroundsubtracted) by the hybridization signal from the flat surface (also withbackground subtracted). This data shows the actual ability both tosynthesize full 20-mer probes and hybridize target DNA to the probes,and thus the actual functional improvement which can be obtained withusing these surfaces for DNA arrays. The porous surfaces yield signalsmany times greater than the flat glass. It has been found that thehybridization signal increases with films spun under decreasing spinspeed. This corresponds to deposition of a thicker layers at slowerspeeds and indicates that fluorescence signal obtained is proportionalto film thickness.

[0167] Thicker films can also be achieved by depositing multiple layers.A multilayer film was created by depositing a layer at 1500 rpm,allowing the film to dry for approximately 15 minutes, and thenrepeating the process. The hybridization signal at the 20 and 40 hourtime points is approximately 2 times higher for the multilayer film thanfor the single layer. Film thickness can be modulated by depositingmultiple layers or by increasing the weight percent of the colloidalsilica in the solution prior to deposition on the glass surface. Thisagain demonstrates the tremendous flexibility of sol-gel deposition andits use for this application.

[0168] It should be noted that the sol-gel layers were formed withoutexplicit control of temperature, humidity, or air flow. Thisdemonstrates the robustness of the process. Controlling these variablesand other environmental factors to refine the properties of the systemis within the capability of one of ordinary skill in the art.

[0169] Ellipsometric measurements on the 500 rpm surface gave an indexof refraction for the film of approximately 1.3. The refractive index ofa porous film is the volume average of the solid phase and the porespace, and thus gives an estimate of the porosity. Using this technique,it is estimated that the above film is approximately 30-40% porous. Filmthickness for the 500 rpm film was measured as 6400+/−300 Å. Combiningthese measurements with the nominal particle size, it is estimated thatthe porous surface will have a maximum surface area of approximately 100times that of a flat surface of comparable lateral dimensions. However,this estimate does not take into account factors such as particlecontacts and TMOS coverage, so the actual area available is smaller thanthis multiple.

[0170] To analyze how much of the surface is being accessed by probesynthesis, an HPLC quantitation analysis of the surface was performed todetermine the site density and this value was compared to thefluorescence signal attained by scanning. For the films spun at 500 rpmwith the TM-40 solution, both procedures give signals which agree withinexperimental error, and indicate that the porous surface hasapproximately 40 times more sites than a flat surface. Furthermore,since the site density and fluorescence agree, quenching is notsignificant or an issue when evaluating surfaces of this density.

[0171] The HPLC results also showed that there are not a significantnumber of fluorescent molecules which are not attached to activatedsynthesis sites, as the only large peaks in the chromatogram were fromthe activated synthesis sites and the internal standard. This is animportant result because molecules getting “stuck” in the matrix couldbe a concern. Given the 40-fold factor improvement (out of a theoretical100-fold maximum), it is clear that the films offer substantialadvantages over flat glass, and also that there is room for furtherimprovement if even more of the surface can be accessed.

[0172] Pore size in these films is estimated as the smallest spacesbetween particles through which a potential target would have todiffuse. For the current system, this is estimated by assuming threesame-sized particles in a triangular formation. In this configuration,the pore size is approximately 0.15 times the particle diameter. Thusfor 22 nm particles, the smallest pores would be on the order of 4 nm.

[0173] The use of larger particles (and thus larger pores) may be oneroute to access more of the surface. An additional advantage ofdepositing films comprised of larger particle sizes, is that there is apotential to use larger target molecules. As will be described infollowing sections, various staining and antibody amplificationtechniques are often used for signal amplification in hybridization tocomplex arrays. These molecules are often much larger than thefluorescein-labeled polynucleotides. For this reason, experiments wereconducted with the larger Snowtex particles before proceeding to complexarrays.

[0174] Using the same procedures as described previously (TMOS,annealing of substrate), films were deposited at 500 and 2500 rpm withSnowtex-20L (referred to as “40 nm”) and Snowtex-ZL (referred to as “70nm”) solutions and hybridized to a target solution (50 nM target,6×SSPE). These films show significant improvement over the flat glass,especially under stringent assay conditions of long hybridization timesat elevated temperature. With these porous surfaces, signal enhancementsof 15 to 45 times that of flat glass were reached, with higher signalsfor smaller particles (higher surface area to volume ratio per particle)and slower spin speeds (thicker films).

[0175] Edge diffraction studies revealed that the porous silica surfacedid not affect light absorbance through the glass substrate or causelight scattering, thus rendering the surface amenable to photochemicalmethods of synthesis. For photochemical methods to be employed on suchsurfaces for the printing of small features, the edge resolution must bevery sharp. The methodology was as described in McGall, et al, supra.The results indicate that there was no discernible distinction (i.e.,diffraction effects) between the flat glass and the porous silica, whichwas verified down to features sizes of 24×24 microns.

[0176] Experiments were conducted to determine if several of the stepsof the original procedure, use of the TMOS and annealing steps, could beeliminated. The primary effect of removing these steps could be aresultant decrease in the stability of the films. This film stabilitywas tested in several ways.

[0177] Films originating from a 10 wt. % ZL solution were deposited andfluoroprime stained as described above. Flat “standard” glass controlswas run as well. The arrays were scanned at intervals and the signal wastracked over the course of several days under harsh assay-typeconditions: MES buffer or 6×SSPE buffer at 45° C. In MES buffer, thefluoreprime stain signal intensity on the porous surfaces decreasedslightly more slowly than the flat glass. The porous films exhibitedsimilar rates of signal decay when compared to the flat glass in 6×SSPEbuffer, The rate signal decrease in the 6×SSPE buffer assay was quickerfor both the porous and flat glasses.

[0178] To further test stability, a functional hybridization assay wasperformed under typical “gene expression” conditions of an overnight(16-18 h) hour hybridization at 45° C. Films were deposited with a 10wt. % ZL solution and a concentrated ZL solution (equivalent to 4-5layers deposited at 10 wt %), yielding films of approximately 1500 and6000 angstroms thick respectively. 20 mer probes were synthesized andhybridized to 10 nM labeled target. The films achieved fluorescencehybridization signals of 6 and 29 times that of the flat glassrespectively, which shows that the signal increase is approximatelylinear with film thickness.

[0179] These films provide examples for determining surfaceaccessibility in the larger pore films. The thickness, index ofrefraction, and light scattering (prior to deposition) analysis arecombined to estimate the surface area of these films. The thinner filmshave a surface area approximately 10 times that of flat glass, and thethicker films are approximately 44 times flat glass. These values arewithin reasonable tolerances of the observed 6-fold and 29-foldrespective increase detected hybridization signals.

[0180] Since the films achieve a signal comparable to their expectedarea, it is expected that chemical coupling is proceeding efficiently.This was tested by the 6-mer coupling method on several films. Flatglass was tested vs. films deposited from 10 wt. % 20L and ZL solutions.Coupling yield is nearly identical to the flat glass on both of theporous substrates.

[0181] Kinetics of hybridization can be determined by real time scanningconfocal microscopy of the to follow the annealing of the probe sequenceto a 5′-fluorescein labeled complementary oligonucleotide target.(Forman, J. E., Walton, I. D., Stern, D., Rava, R. P., Trulson, M. O″,Thermodynamics of Duplex Formation and Mismatch DiscriminationOnphotolithographically Synthesized Oligonucleotide Arrays Acs SymposiumSeries 682:206-228,1998)

[0182] With a 1500 Angstom porous glass layer of derived from the 70-100nm particles, hybridization equilibrium at room temperature is typicallyreached in approximately 2 h. The time to reach equilibrium increased to3-5 h for a 3000 Angstom layer as compared to 40-60 minutes for standardflat surface.

[0183] The substrates provided herein can be used with any type of arrayformation pattern. It can be readily appreciated that by varying theexperimental conditions such as viscosity, thickness of layers, size ofthe colloidal particles, aging time and pH of the colloidal mixture, onecan control layer thickness, porosity, morphology, and surface chemistryin order to optimize the system. For example, raising the viscosity(such as by adding less water) or lowering the spin speed can increasefilm thickness. Depositing multiple layers of the same thickness ordifferent layers of varying thickness would also result in porous layersof the desired thickness. Additionally, other properties such asparticle size could be varied on different layers. Controlling theenvironment, such as humidity, partial pressures of other solvents (suchas methanol), air flow, temperature can also lead to porous glass ofdesired pore size and layer thickness.

[0184] Since varying the colloid size changes pore size, one can obtainthe desired pore size by appropriately formulating the colloidal mixtureof proper particle size initially. Colloid aggregation can be controlledby varying the “aging” time and pH before deposition. Other ceramiccolloids, such as titania or alumina, which may have different surfacechemistry and/or aggregation properties can also be used to preparecolloids of desired composition and size. Additionally, surfacechemistry of the colloidal particles can be controlled for example, bysilanating the colloidal particles before deposition, in order to tailorthe chemistry of the final film.

[0185] Optical scattering can be reduced by using refractiveindex-matched fluids. These fluids reduce optical scattering duringpatterning and scanning of the chips. Water has a refractive index of1.33, whereas that of glass is typically 1.45-1.52. An example of anindex-matched fluid is an aqueous solution of 64% by weight sucrosewhich has an index of 1.45, or dioxane with index of 1.42.

[0186] The preparation and synthesis of porous glass substrates isamenable to scale up. Preliminary studies were performed on 2×3 slides.Current manufacturing of “real” GeneChip® arrays requires synthesis tobe performed on a 5″×5″ fused silica wafer and synthesized on anAFFYMETRIX MOS synthesizer. Moving to this scale posed several newchallenges for porous surfaces, all which have been met so far.

[0187] Preliminary evaluation of wafer uniformity showed no difficultiesin scaling up. The wafers can be coated uniformly by spin coating andmost tests that were performed in initial evaluations of the poroussurfaces were also used to monitor uniformity across the wafer. Thesetests involve functional assessment of chip performance as a function oflocation on the wafer.

[0188] There are many mechanical and chemical demands during wafer scalesynthesis of DNA arrays. A synthesis cycle is comprised of chemicaldelivery of reagents on the MOS, followed by removal of the wafer thenalignment on a photolysis station. Typical GeneChip® array synthesisinvolves 70-80 such cycles, consuming up to 18 hrs. of processing andhandling, followed then by deprotection, dicing the wafer intoindividual arrays, and packaging into cartridges. Wafers coated withfilms comprised of colloidal silica particles survived these varioussteps and showed higher functional performance that flat glass.

[0189] The porous silica substrates of the present invention alsopossess excellent array feature characteristics. Checkerboard patternsthat contain 400×400 micron features as well as the 24 micron featurespresent on the Human 6800 arrays exhibited no defects resulting lightscattering- or diffraction-related difficulties during either thephotolithographic synthesis or scanning microscopy on the experiments asdescribed herein.

[0190] Porous inorganic colloidal silica particle size ranging from 12nm-greater than 100 nm was deposited on a glass surface. The glasssurface was made of but limited to either soda lime, borofloat or fusedsilica glass. The resultant pores from such particles are in the 2-40 nmrange. Layers from 0.1 micron or 2 micron were actively investigated.The layers could be thicker or thinner.

[0191] The porous silica substrates of the present invention allow thesynthesis of high-density 3-dimensional arrays. High-density arrays wereprepared on layers of silica particles of approximately 0.25-0.3 micronsthickness which are prepared from a 20% (by weight) colloidal silicasolution. Two representative complex high-density DNA arrays will bediscussed in detail here to exemplify the capabilities of the presentporous glass substrates: one representing a sequence analysis (diseasemanagement) type array and one representing a gene expression array. A0.3 micron porous surface was selected for synthesis of GeneChip® arraysfor evaluation in both disease management and gene expression typeassays.

[0192] Sequence Analysis Array

[0193] Sequence analysis or disease management arrays are typically butnot limited to “tiling type-arrays.” Assay times are relatively shortbecause target concentrations are not limited. The target may be labeledwith either fluorescein for direct detection or may be labeled withbiotin for detection via signal amplification. A GeneChip® test arraycontaining an HIV sequence was synthesized on a porous glass substrateand a representative HIV assay was performed. The array on this testvehicle is comprised of probes representing the HIV protease and reversetranscriptase genes. The sequence analysis assay was performed onfragmented fluorescein-labeled HIV cRNA target. The surface was scannedat regular intervals and was approaching equilibrium at 6 hours, atwhich time there was a 4-6 fold increase in hybridization signalintensity over flat glass. Furthermore, at this time the base calldiscrimination on the porous surface was comparable to the flat surface.The assay time is somewhat longer than typical assays of this type, butas a practical matter, these longer assay times are not material. Thisassay further verifies that fragmented RNA target can indeed access theprobe sites within the porous matrix. Large RNA fragments or reagents donot get trapped within the matrix as there is no increase backgroundsignal and there is no reduced dicrimination by probes for target.

[0194] Gene Expression Monitoring

[0195] A Human 6800 array (HuF1) was synthesized (24 micron features;16-20 probe pairs /gene) on a 0.3 micron porous silica substrate coatedwith either BIS or GPS silane (BIS[2-hydroxyethyl]-3-aminopropyl-triethoxysilane or 3-glycidoxylpropyltrimethoxy silane respectively) (Gelest, Tullytown, Pa.) and compared tothe appropriately silanated flat glass control. The GPS silane can bedeposited in the vapor or solution phase followed by ring opening withacid. Standard quality control assay, which involves hybridization offour biotinylated control gene transcripts as well as twelvebiotinylated polynucleotide targets in 6×SSPE buffer for 16 h at 45° C.followed by staining with streptavidin-phycoerythryn complex (SAPE)(Molecular Probes, Eugene Oreg.), showed that the BIS and GPS silanesyield similar hybridization results on a given surface type. A 1-3-foldenhancement in hybridization signal intensity and average difference(perfect match minus mismatch divided by the total number of probes in agene) was observed on the porous silica substrates relative to the flatglass with the control gene probe pairs (RNA target). The porous silicasubstrate surface exhibited a 7-fold increase in the average differencedata with the DNA target polynucleotides.

[0196] Additional assays involved detection of 9 biotinylated-controlgenes spiked into the hybridization mixture that contained a backgroundof complex labeled human RNA target. Assays were conducted and materialswere obtained as described in The Affymetrix Gene Chip ExpressionAnalysis Manual, 1999. Assays were performed in MES buffer at 45° C. for16 h followed by staining with SAPE. These assays show that the poroussilica substrate results in an enhancement in signal of four to six foldwith respect to the flat glass surface. Similar values were obtained bylooking at the average difference for the control genes. Scatter plotscomparing all the genes on the wafer reveal that this trend holds withall the probe sets on the surface and indicate uniform surface responseto target.

[0197] Further signal amplification by using a second staining step asis commonly done in complex gene expression analysis assays involvestreating the SAPE stained surface with an antibody (IgG) followed by asecond round of SAPE staining. Again this led to another six foldincrease in signal on both surfaces and a four to six fold increase insignal on the porous silica substrates relative to the flat glass. Thediscrimination on both surfaces is the same. Furthermore, thediscrimination on the porous surfaces can be additionally improved byemploying more stringent wash conditions.

[0198] The results thus show that gene expression monitoroing typeassays on porous silica substrates have yielded very high signalintensities under the standard conditions without any assayoptimization. Assay optomization which is underway may improve thisfurther. This is significant because, it is generally known that moresensitivity is needed in expression monitoring assays. Additionally,because the signal is much stronger after the first stain, it may bepossible to eliminate further staining/signal amplification or cut backthe time necessary to run the assay. No increase in nonspecific bindinghas been observed. Clearly the pores generated between the particlesenable diffusion of target, label (SAPE) and antibodies to access thesites. Thus, the porous silica substrates of the present inventionappear to be superior to the traditional flat glass substrates in manyimportant aspects and appear to provide many advantages in high-densityarray synthesis and assays.

Example 2

[0199] Particle Templating

[0200] The approach of depositing colloidal silica to form a porouslayer for DNA arrays shows a tremendous potential for using porousinorganic layers as supports for biosensing devices. Furthermore, itdemonstrates a very simple and reproducible technique that can beeffective. However, this technique can be further improved with atemplating process to further control pore size and porosity, two keyaspects of the film morphology. Control of pore size is desirable for atleast two aspects of hybridization. First of all, larger pores allow forlarger molecules to penetrate the matrix. Thus, arbitrary targets can beselected and the morphology of the porous layer (surface) altered tomatch. Second it is known from literature that flow in a porousstructure is affected by pore size. Larger pores provide the potentialadvantage of increasing flow rates and thus decreasing hybridization(processing) times, thereby boosting assay performance.

[0201] In the technique described, pore size and porosity are controlledby co-depositing sacrificial organic particles, such as polymer (e.g.polymer latex) spheres, with the silica particles, and then burning outthe organic material at high temperatures or otherwise removing it.Templating using a sacrificial material is not a new technique initself. Studies have been conducted where latex particles served as poretemplates in gelled silica networks formed from alkoxysilanes. Thecurrent approach is novel at least because only particles are used forboth the template and the silica matrix that remains after pyrolysis. Asis described in the following section, simple modificiations of thesize, charge, and concentration of the latex can provide a wide range offilm morphologies. In typical deposition with alkoxysilanes, morecomplicated changes in the chemistry and processing are necessary toalter the morphology. Additionally, an important advantage of thisapproach for the application of polymer synthesis and hybridization isthat the voids inherent in the particle system ensure open porosity,whereas films formed from alkoxysilanes with templating can oftenresults in closed pores. The open porosity results in at least somepores being connected to each other, thus allowing fluids to pass intothe porous layer of the substrate through the free surface of thesubstrate.

[0202] The polymer to be co-deposited with the silica can be anysuitable polymer that achieves the objective of providing the desirableporous structure. One of ordinary skill in the art would understand thatseveral such polymers are available for the present purposes, forexample, polystyrene latex can be used.

[0203] By the term “co-depositing”, it is meant here that the silica andthe polymer need not be presented simultaneously so long as the polymerand the silica occupy the surface of the substrate to provide thedesired porosity upon the removal of the polymer.

[0204]FIG. 3a is a simplified cross section of a portion of a substrate140 being processed to form a porous region according to a templatingmethod. Templating particles 142 have been mixed with smaller silicaparticles 144 (shown without cross hatching for purposes of clarity) andapplied to an underlying substrate or support region 104. It is believedthat the silica particles coat the templating particles (e.g., latexspheres). After the silica and templating particles are applied to theunderlying substrate, the templating material can be burned off, leavingbehind a matrix of the silica particles. The particles can be of thesame or different size. In one embodiment, the small silica particlesfurther enhance the effective area of the porous layer. Porosity andpore size are increased by voids left from the removal of the templatingmaterial. In other embodiments, the remaining silica particle can besintered or annealed to strengthen the remaining matrix.

[0205]FIG. 3b is a simplified cross section of the portion of a poroussubstrate 141 after the templating particles have been removed, such asin a burn-off process. The burn-off process has removed the templatingparticles (compare FIG. 3b, reference numeral 142) to leave behind amatrix of silica particles 144. The voids left by the templatingparticles provide increased effective surface area for the poroussubstrate. The silica particles may be further processed, such as in anannealing process, to further densify the matrix 146 of silicaparticles.

[0206]FIG. 4 is a simplified cross section of a portion of a substrate150 being processed to form a porous layer according to anotherembodiment of the present invention. Templating particles 152 are mixedwith silica particles 154 and applied to the underlying substrate 104,such as by spin coating. The templating particles are then removed, asdiscussed above in reference to FIGS. 3a and 3 b, leaving behind amatrix of silica particles. As discussed above in relation to FIG. 1,the section viewed represents particles of essentially the same sizethat intersect the section plane. The various diameters shown in thefigure represent sections of the particles, some of which are notsectioned through their center. It is understood that, generally, eachparticle touches several other particles, thus when the templatingmaterial is removed a silica matrix forms. It is further understood thatparticles other than silica could be used to form the resulting matrix.

[0207] Experimental

[0208] Polystyrene- latex dispersions (hereinafter referred to as“latex” or “latex particles”) were purchased from INTERFACIAL DYNAMICSCORPORATION of Portland, Oreg. The solutions are stabilized either bynegatively charged sulfate groups on the surface or positive amidinegroups. Particles with other surface groups could also be used.

[0209] In a typical film deposition, the latex and silica solutions aremixed and diluted with pure water to the desired concentrations.Exemplary concentrations include 10:1 by volume latex to 1:1 by volumelatex. The solution was then filtered through a 450 nm syringe filterand dispensed onto either glass or a silicon wafer as describedpreviously. Thick yet homogeneous films could be obtained at spin speedsof 1000 rpm and greater (70 second spin time). Thicker films can beobtained by using slower spin speeds, more concentrated solutions, ormultiple depositions.

[0210] Following spin coating, the films were annealed to removepolymer. Typical conditions were to ramp 2° C./minute to 400° C., dwell4 hours, then cool to 20° C. at rate of 20° C./minute. Followingannealing, a final cleaning step was applied to some samples. This stepconsisted of immersing the annealed substrate in either piranha solution(30 minutes) and/or etching in 1M sodium hydroxide at 70° C. (3minutes).

[0211] Results

[0212] Atomic force microscopy images of co-deposited 40 nm silica and60 nm latex particles have been taken before annealing in “phasecontrast mode,” which shows the distribution of the two types ofparticles. From the images it can be observed that the deposition isrelatively homogeneous. In an ideal distribution, silica and latexparticles would alternate in an “array” manner. While the distributiondoes not reach this level of homogeneity, extensive inter-mingling ofthe phases can be observed.

[0213] The uniqueness and flexibility of using particles to create thefinal films was demonstrated by two examples. In a first example, 7 nmsilica particles were mixed in solution with 100 nm, positively chargedlatex particles. The charge difference causes the relatively smallsilica particles to coat the larger latex particles. This sample wasdipcoated by withdrawing slowly from solution with the use a variablespeed motor. Following annealing, large pores on the order of 100 nmwere left behind, with a relatively dense matrix formed from theremaining silica.

[0214] Very different structures were created by mixing same-sized,same-charge particles, such as negatively charged 40 nm latex and silicaparticles of the same size. As silica is typically negatively charged inthis environment, the latex and silica do not substantially aggregate. Apotential advantage of these films is that the pores left behind by theannealed latex are better connected to each other by the passagesbetween the larger silica particles than in non-templated films. Incomparison to the films created with the larger particles and notemplating (such as SNOWTEX-ZL), the templated films have pores whichare as large (or larger) with surface area that is as high (or higher).

[0215] The techniques of light scattering, ellipsometry, and thicknessprofiling were combined to characterize and estimate the surface areaper unit thickness for the templated films. The statistics shown inTable 2 compare a templated film made from 20 nm Latex/SNOWTEX-50 2:1v/v film with a pure silica film made from SNOWTEX-ZL. This tabledemonstrates the potential benefit of templated films. Several factorsshould be noted. First of all, for approximately the same filmthickness, the templated film has nearly 3 times the area. Second, thepores in the templated film are actually as large or larger than thepure silica film. The pores in the film made with SNOTEX-ZL (70-100 nmparticles) are on the order of 15 nm, whereas the templated film formedwith latex particles has 20 nm pores; thus, even though the startingparticle size of the pure silica film is approximately 5 times largerthan the starting particle size of the templated film, the templatedfilm provides pores that are greater than about 30% larger. Finally, theporosity is much higher, which gives much more room for a potentialtarget to diffuse into the matrix. Thus, the templated film provides amuch more efficient substrate for some applications. TABLE 2 Comparisonof Pure and Templated Films Templated 20 nm silica (2:1 Pure 70 nmsilica v/v latex:silica) Thickness 2200 2500 Index 1.37 1.16 Porosity20% 65% Area factor 14.5 35.4 Area/thickness 6.6 14.2 (per 0.1 micron)

[0216] Full 20 mer probes were synthesized on the films described inTable 2. A kinetic scan of the adsorbed target vs. time was performed.The conditions used were room temperature, 10 nM target, flow of 4ml/min, MES buffer. The kinetic scan demonstrates the advantage of usingtemplated films. For a film of similar thickness, a higher signal isreached. The pure non-templated film reaches a signal 14 times that ofthe flat glass, where the templated glass reaches a signal 40 times thatof flat glass. These signals agree well with the area factors estimatedin Table 2. Additionally, the templated film reaches equilibrium morequickly (3 hrs vs. 5 hrs.).

Example 3

[0217] Etched Sodium Borosilicate Glass

[0218] Glass samples of sodium borosilicate glass, suitable for use in aVYCOR™ process, were obtained from Dow CORNING, in the form of150×150×0.7 mm sheets. The specifications are for a composition of 67.4%SiO₂, 25.7% B₂O₃, 6.9% Na₂O (by weight), but the precise ratios ofcomponents may vary within known ranges. These sheets where then dicedinto 10×10 mm pieces for testing. The test pieces were annealed at 650°C. for 4 hours to separate the glass into regions of a sodium andboron-rich phase and regions of a silica-rich phase. The samples werethen placed in 4% rich phase, preferentially leaving a matrix of thesilica-rich phase.After leaching, the samples were soaked in methanolfor approximately 15 minutes. The glass was then cleaned in a sodiumhydroxide solution (20 g/liter) that to dissolve silica that mightremain in the pores following the leaching step. Finally, the glass wasagain soaked in methanol for 15 minutes, and allowed to dry at roomtemperature.

[0219] Immediately before silanation, the test pieces were placed insodium hydroxide solution (20 g/liter) at 70° C. for 3 minutes, followedby water for 15 minutes. This step was used to further insure that thesurface would be covered with hydroxyl groups that may have been removedduring the HF treatment.

[0220] The films are silanated with the same procedures described aboveas in the case of sol-gel porous silica. Fluorescent staining wasperformed with 5 mM concentrations of fluorescein, in a manner similarto that described above in the case of sol-gel silica. Exceptions arethat the test pieces were mounted on 2×3 in slides usingroom-temperature vulcanizing (“RTV”) silicone glue available from DOWCORNING, and a 20 mil fluoropolymer tape (Polyken) was used as a spacer.This tape showed minimal degradation by solvents over the period forfluorescent staining.

[0221] Results

[0222] Cross sectional scanning electron microscopy reveals an etchedsurface layer of approximately 70 microns. It is not clear whether theporosity is interconnected through the depth of the entire layer,although it can be assumed since the etch solution must penetrate intothe matrix. Large pores can be observed, on the order of 1000 Å andlarger, although rigorous measurements of pore size were not made. Usingthese measurements, it was estimated that a layer of 70 microns thickcould increase the surface area by as much as 400 times over the flatarea of the glass.

[0223] Staining the surface with fluorescene, as described above,revealed that the etched porous surface yielded a fluorescence signalgain 200 times that of flat glass. Additionally, for the processexamined, the etched porous surface actually had a lower background,that is as a percent of the total signal, resulting in an overallincrease in the number of sites of over 300 times the flat glass aftercorrection for background.

[0224] Additionally, site density was examined using the HPLCquantitation procedure. The HPLC analysis confirmed a factor of 200increase in accessible sites. However, the chromatogram from the HPLCalso showed that there was a significant fraction of fluorescentmolecules removed from the surface which were not attached to activatedsites (i.e. fluorescent molecules not attached to C₃ spacers molecules).These may be molecules which were “stuck” in the matrix during thestaining procedure. This result differs from the sol-gel system in whichall the observed signal was from covalently attached molecules. Thistrapping of molecules within the matrix is most likely due to theextremely thick surface layer, and using shorter etch time to obtain athinner layer could decrease the portion of molecules not attached tosynthesis sites.

[0225] The above experiments show that the etched borosilicate glasslayer of the above-described composition holds significant promise foruse as a substrate in a variety of polymer applications, includinghigh-density polymer synthesis and assays. When the polymer is apolynucleotide or a nucleic acid, a number of assays comprisinghybridization can be performed, as pointed out in Detailed Descriptionabove. When the polymer is a polypeptide or a protein, their kineticfunctions and antigen-antibody reactions, can be performed. Theborosilicate system can be tailored to suit the application by simplymodifying the annealing time (e.g., longer annealing increases poresize) and etching time (e.g., longer etch creates thicker layer).

What is claimed is:
 1. A porous substrate comprising: a support region;and a porous region on the support region, the porous region beingprimarily inorganic and having a surface capable of forming a polymerarray thereon, the porous region comprising pores of a pore size ofabout 2 nm-500 nm, a porosity of about 10-90%, and a thickness of about0.01 μm to about 70 μm.
 2. The porous substrate of claim 1, wherein theporous region is formed by an additive method.
 3. The porous substrateof claim 2 wherein the additive method includes the application ofcolloidal silica on the support region.
 4. The porous substrate of claim2 wherein the additive method includes the application of alkoxysilaneon the support region.
 5. The porous substrate of claim 1 wherein theporous region comprises silica.
 6. The porous substrate of claim 5wherein the porous region further comprises organic polymer of less thanor equal to about 10% mole fraction.
 7. The porous substrate of claim 5,wherein the porous region comprises a plurality of pores, each of theplurality of pores having a size of from about 2 to about 100 nm.
 8. Theporous substrate of claim 5, wherein the porous region comprises aplurality of pores, each of the plurality of pores having a size of fromabout 2 to about 50 nm.
 9. The porous substrate of claim 1, wherein theporous region has a porosity of from about 20 -80%.
 10. The poroussubstrate of claim 1, wherein the porous region has a porosity of fromabout 50-70%.
 11. The porous substrate of claim 5, wherein the porousregion comprises a plurality of particles, each of the plurality ofparticles having a size from about 5-500 nm.
 12. The porous substrate ofclaim 5, wherein the porous region comprises a plurality of particles,each of the plurality of particles having a size from about 5-200 nm.13. The porous substrate of claim 5, wherein the porous region comprisesa plurality of particles, each of the plurality of particles having asize from about 70-100 nm.
 14. The porous substrate of claim 2 whereinthe porous region has a thickness from about 0.1-1 microns.
 15. Theporous substrate of claim 2, wherein the porous region has a thicknessof from about 0.1 μm to about 0.5 μm.
 16. The porous substrate of claim2, wherein the porous region has a thickness of from about 1 μm to about20 μm.
 17. The porous substrate of claim 6, wherein the organic polymercoats silica particles of the porous region.
 18. The porous substrate ofclaim 5, wherein the porous region is silylated with a silyating agent.19. The porous substrate of claim 18, wherein the silylating agent isselected from the group consisting ofN,N-bis(hydroxyethylaminopropyl)triethoxysilane and glycidoxypropyltrimethoxy silane.
 20. The porous substrate of claim 2, wherein theporous region is formed by codepositing an organic template materialwith silica, followed by removing the organic template material.
 21. Theporous substrate of claim 20 wherein the organic template materialcomprises particles of about 10-100 nm and the silica comprisesparticles of about 7-100 nm.
 22. The porous substrate of claim 21wherein an organic template particle size is about equal to a silicaparticle size.
 23. The porous substrate of claim 21 wherein a silicaparticle size is less than or equal to about ⅔ an organic templateparticle size.
 24. The porous substrate of claim 21 wherein a silicaparticle size is less than about 10% of an organic template particlesize.
 25. The porous substrate of claim 20 wherein the organic templatematerial is deposited in a volume ratio to the silica of about 10:1 to1:10.
 26. The porous substrate of claim 20 wherein the organic templatematerial is removed using a baking process at a temperature of aboveabout 150° C.
 27. The porous substrate of claim 26 wherein the silica isdensified using an annealing process.
 28. The porous substrate of claim20 wherein the porous region has an effective surface area of about15-40 times a flat substrate with an equivalent two dimensionalstructure.
 29. The porous substrate of claim 1 wherein the porous regionis formed by a subtractive method.
 30. The porous substrate of claim 20,wherein the organic template polymer is a latex polymer.
 31. The poroussubstrate of claim 29 wherein the porous substrate comprisesphase-separable glass, a surface portion of the phase-separable glassbeing treated to form the porous layer.
 32. The porous substrate ofclaim 31 wherein the phase-separable glass comprises a sodiumborosilicate glass.
 33. The porous substrate of claim 32 wherein thesodium borosilicate glass has been annealed and leached to provide theporous layer having a thickness of about 70 microns and comprised of aplurality of pores, at least some of the plurality of pores having apore size greater than about 1000 Å.
 34. The porous substrate of claim29 wherein the porous region has an effective surface area of about50-400 times a flat substrate with an equivalent two dimensionalstructure.
 35. The substrate of claim 1, further comprising a highdensity array of nucleic acids immobilized on the surface.
 36. A poroussubstrate comprising: a support region; and a porous region on thesupport region, said porous region of about 0.1-0.5 microns thick,wherein the porous layer comprises an unsintered matrix formed from atleast colloidal silica having a particle size of about 70-100 microns,the unsintered matrix defining at least a plurality of open pores havinga pore size of about 10-20 nm, and wherein the porous layer has aporosity of about 10-90%.
 37. A method of forming a porous substrate,the method comprising: providing a substrate material comprising asurface; dipping the substrate material in a solution includingcolloidal silica and a carrier, the colloidal silica having a particlesize of about 12-100 nm; and withdrawing the substrate material toprovide an unsintered porous layer having a thickness of about 0.1-1microns and a porosity of about 10-90% on the substrate material.
 38. Amethod of forming a porous substrate, the method comprising: providing asubstrate material comprising a surface; applying a solution includingcolloidal silica and a carrier to the surface of the substrate material,the colloidal silica having a particle size of about 12-100 nm; spinningthe substrate material and the applied solution to achieve a spun layeron the substrate material; and removing the carrier from the spun layerto provide an unsintered porous layer having a thickness of about 0.1-1microns and a porosity of about 10-90% on the substrate material.
 39. Amethod of forming a porous substrate comprising different monomersequences, the method comprising: immobilizing different monomersequences on a porous substrate of claim
 1. 40. A method of synthesizingpolymers on a porous substrate, the method comprising: a) generating apattern of light and dark areas by selectively irradiating at least afirst area of a surface of a porous substrate of claim 1, said surfacecomprising immobilized monomers on said surface, said monomers coupledto a photoremovable protective group, without irradiating at least asecond area of said surface, to remove said protective group from saidmonomers in said first area; b) simultaneously contacting said firstarea and said second area of said surface with a first monomer to couplesaid first monomer to said immobilized monomers in said first area, andnot in said second area, said first monomer having said photoremovableprotective group; c) generating another pattern of light and dark areasby selectively irradiating with light at least a part of said first areaof said surface and at least a part of said second area to remove saidprotective group in said at least a part of said first area and said atleast a part of said second area; d) simultaneously contacting saidfirst area and said second area of said surface with a second monomer tocouple said second monomer to said immobilized monomers in at least apart of said first area and at least a part of said second area; and e)performing additional irradiating and monomer contacting and couplingsteps so that a matrix array of different polymers is formed on saidsurface, whereby said different polymers have sequences and locations onsaid surface defined by the patterns of light and dark areas formedduring the irradiating steps and the monomers coupled in said contactingsteps.
 41. The method of claim 40, wherein the monomers are selectedfrom the group consisting of: nucleotides, amino acids, andmonosaccharides.
 42. The method of claim 40, wherein the substrate haslinker molecules on its surface.
 43. A method of forming polymers havingdifferent monomer sequences on a porous substrate, the methodcomprising: providing a porous substrate of claim 1 comprising a linkermolecule layer thereon, said linker molecule layer comprising a linkermolecule and a protective group; applying a barrier layer overlying saidlinker molecule layer, said applying step forming selected exposedregions of said linker molecule layer; exposing said selected exposedregions of said linker molecule layer to a deprotecting agent to removethe protective group; and coupling selected monomers to form selectedpolymers on the substrate.
 44. The method of claim 43, wherein thedeprotection agent is in the vapor phase.
 45. The method of claim 43,wherein said deprotection agent is an acid.
 46. The method of claim 45,wherein the acid is selected from a group consisting of trichloroaceticacid, dichloroacetic acid, and HCl.
 47. The method of claim 43, whereinthe monomers are selected from the group consisting of nucleotides,amino acids, and monosaccharides.
 48. A method for detecting a nucleicacid sequence, the method comprising: (a) providing an array of nucleicacids bound to the porous substrate of claim 1; (b) contacting the arrayof nucleic acids with at least one labeled nucleic acid comprising asequence substantially complementary to a nucleic acid of said array,and (c) detecting hybridization at least the labeled complementarynucleic acid to nucleic acids of said array.